[Show abstract][Hide abstract] ABSTRACT: Members of the receptor tyrosine kinase family (RTK) have been shown to be present in the nucleus of cells; however, the mechanisms
underlying their trafficking to the nucleus, and their relevance once there are poorly understood. In the present study, we
focus on the RTK ErbB3 and elucidate the mechanisms regulating its trafficking. We show that heregulin-stimulation induces
trafficking of phosphorylated ErbB3 from the plasma membrane to the nucleus via a clathrin-independent mechanism. Nuclear
import of ErbB3 occurs via importin β1, which drives the receptor through the nuclear pore complex. In the nucleus, ErbB3
interacts with transcription complexes, and thereby has a role in transcriptional regulation. Our results also demonstrate
that ErbB3s nuclear localization is transient as it is exported out of the nucleus by the nuclear receptor protein crm-1.
Analysis of normal, regenerating tissues, and tumors showed that ErbB3 nuclear translocation is a common event in proliferating
Preview · Article · Dec 2015 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: Cholestasis is a common complication in liver diseases that triggers a proliferative response of the biliary tree. Bile duct ligation (BDL) is a frequently used model of cholestasis in rodents. To determine which changes occur in the 3D-architecture of the interlobular biliary duct (BD) during cholestasis, we used 3D-confocal imaging, surface reconstructions and automated image quantification covering a period up to 28 days after BDL. We show a highly reproducible sequence of interlobular duct remodeling, where cholangiocyte proliferation initially causes corrugation of the luminal duct surface, leading to an approximately 5-fold increase in surface area. This is analogous to the function of villi in the intestine or sulci in the brain, where an expansion of area is achieved within a restricted volume. The increase in surface area is further enhanced by duct branching, branch elongation and finally loop formation through self-joining, whereby an initially relatively sparse mesh surrounding the portal vein becomes 5-fold denser through elongation, corrugation and ramification. The number of connections between BD and the lobular bile canalicular network by the canals of Hering decreases proportionally to the increase in BD length, suggesting that no novel connections are established. The diameter of the interlobular BD remains constant after BDL, a response qualitatively distinct from that of large bile ducts which rather tend to enlarge their diameters. Therefore, volume enhancement is only due to net elongation of the ducts. Since curvature and tortuosity of the BD are unaltered this enlargement of the biliary tree is caused by branching and not by convolution. In conclusion, BDL causes adaptive remodeling that aims at optimizing the intraluminal surface area by corrugation and branching. This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: The comprehensive transcriptomic analysis of clinically annotated human tissue has found widespread use in oncology, cell biology, immunology, and toxicology. In cancer research, microarray-based gene expression profiling has successfully been applied to subclassify disease entities, predict therapy response, and identify cellular mechanisms. Public accessibility of raw data, together with corresponding information on clinicopathological parameters, offers the opportunity to reuse previously analyzed data and to gain statistical power by combining multiple datasets. However, results and conclusions obviously depend on the reliability of the available information. Here, we propose gene expression-based methods for identifying sample misannotations in public transcriptomic datasets. Sample mix-up can be detected by a classifier that differentiates between samples from male and female patients. Correlation analysis identifies multiple measurements of material from the same sample. The analysis of 45 datasets (including 4913 patients) revealed that erroneous sample annotation, affecting 40 % of the analyzed datasets, may be a more widespread phenomenon than previously thought. Removal of erroneously labelled samples may influence the results of the statistical evaluation in some datasets. Our methods may help to identify individual datasets that contain numerous discrepancies and could be routinely included into the statistical analysis of clinical gene expression data.
No preview · Article · Nov 2015 · Archives of Toxicology
[Show abstract][Hide abstract] ABSTRACT: Arsenic is a human carcinogen that occurs ubiquitously in soil and water. Based on epidemiological studies, a benchmark dose (lower/higher bound estimate) between 0.3 and 8 μg/kg bw/day was estimated to cause a 1 % increased risk of lung, skin and bladder cancer. A recently published study by EFSA on dietary exposure to inorganic arsenic in the European population reported 95th percentiles (lower bound min to upper bound max) for different age groups in the same range as the benchmark dose. For toddlers, a highly exposed group, the highest values ranged between 0.61 and 2.09 µg arsenic/kg bw/day. For all other age classes, the margin of exposure is also small. This scenario calls for regulatory action to reduce arsenic exposure. One priority measure should be to reduce arsenic in food categories that contribute most to exposure. In the EFSA study the food categories 'milk and dairy products,' 'drinking water' and 'food for infants' represent major sources of inorganic arsenic for infants and also rice is an important source. Long-term strategies are required to reduce inorganic arsenic in these food groups. The reduced consumption of rice and rice products which has been recommended may be helpful for a minority of individuals consuming unusually high amounts of rice. However, it is only of limited value for the general European population, because the food categories 'grain-based processed products (non rice-based)' or 'milk and dairy products' contribute more to the exposure with inorganic arsenic than the food category 'rice.' A balanced regulatory activity focusing on the most relevant food categories is required. In conclusion, exposure to inorganic arsenic represents a risk to the health of the European population, particularly to young children. Regulatory measures to reduce exposure are urgently required.
Full-text · Article · Nov 2015 · Archive für Toxikologie
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND:
Tumor cell migration is a prerequisite for metastasis formation. The role of the actin-modulating protein, gelsolin, in metastasis is controversial, as previous studies have reported associations with both worse and better prognosis.
MATERIALS AND METHODS:
We analysed the association of gelsolin mRNA levels with metastasis-free survival in three cohorts (n=766) of patients with node-negative breast cancer. To determine its effect on migration, gelsolin expression was down-regulated as well as overexpressed in breast cancer cell lines.
Higher gelsolin expression correlated with lower tumor stage and grade, and slower cell proliferation, and was associated with longer metastasis-free survival (hazard ratio (HR)=0.60, p<0.001) in patients with estrogen receptor-positive (ER(+)) erb-b2 receptor tyrosine kinase 2-negative (HER2(-)) tumors. Conversely, the opposite association was observed in those with ER(-)HER(-) tumors (HR=1.95, p=0.014). Down-regulation of gelsolin using siRNA in MCF-7 and MDA-MB-468 cells increased cell migration, whereas overexpression had the opposite effect.
High gelsolin levels are associated with better prognosis in ER(+)HER2(-) breast cancer and a reduction in tumor cell migration.
No preview · Article · Oct 2015 · Anticancer research
[Show abstract][Hide abstract] ABSTRACT: Efficient protocols to differentiate human pluripotent stem cells to various tissues in combination with -omics technologies opened up new horizons for in vitro toxicity testing of potential drugs. To provide a solid scientific basis for such assays, it will be important to gain quantitative information on the time course of development and on the underlying regulatory mechanisms by systems biology approaches. Two assays have therefore been tuned here for these requirements. In the UKK test system, human embryonic stem cells (hESC) (or other pluripotent cells) are left to spontaneously differentiate for 14 days in embryoid bodies, to allow generation of cells of all three germ layers. This system recapitulates key steps of early human embryonic development, and it can predict human-specific early embryonic toxicity/teratogenicity, if cells are exposed to chemicals during differentiation. The UKN1 test system is based on hESC differentiating to a population of neuroectodermal progenitor (NEP) cells for 6 days. This system recapitulates early neural development and predicts early developmental neurotoxicity and epigenetic changes triggered by chemicals. Both systems, in combination with transcriptome microarray studies, are suitable for identifying toxicity biomarkers. Moreover, they may be used in combination to generate input data for systems biology analysis. These test systems have advantages over the traditional toxicological studies requiring large amounts of animals. The test systems may contribute to a reduction of the costs for drug development and chemical safety evaluation. Their combination sheds light especially on compounds that may influence neurodevelopment specifically.
No preview · Article · Jun 2015 · Journal of Visualized Experiments
[Show abstract][Hide abstract] ABSTRACT: Liver disease is often associated with altered tissue microarchitecture. However, robust techniques for immunostaining, 3D reconstruction and quantification still represent a bottleneck. To bridge this gap, we have established protocols of antibody combinations for immunostaining, confocal imaging, three-dimensional reconstruction of 100 µm thick tissue blocks, and quantification of key architectural features in the liver. For the simultaneous visualization of bile canaliculi, sinusoidal endothelial cells and central veins as well as DNA, dipeptidyl-peptidase IV/CD26, donkey anti-mouse IgG, glutamine synthetase (GS) antibodies as well as DAPI were applied, respectively. In the second protocol -where the S-phase positive and negative nuclei (BrdU positive versus DAPI), sinusoidal endothelial cells and GS were co-stained. The protocols include three-dimensional reconstruction of the bile canalicular (BC) and sinusoidal networks from the same tissue block, robust capture of position, size and shape of individual hepatocytes as well as entire lobules from the same tissue block. Image processing and analysis were performed by freely available software-TiQuant-(www.msysbio.com/tiquant) developed in the group of Dirk Drasdo. The routinely quantified parameters in adult mice (C57Bl6/N) are the hepatocyte volume (5128.3 ± 837.8 µm3), the fraction of the hepatocyte surface in contact with the sinusoids (22.1 ± 4.8%) and neighboring hepatocytes (67.4 ± 6.7%) as well as bile canaliculi (9.9 ± 3.8%). Furthermore, fundamental parameters can be quantified in the sinusoidal network such as the branching angle (32.5 ± 11.2 °), the radius of the sinusoids (4.8 ± 2.25 µm), the length of intersection branch (23.93 ± 5.9 µm), the number of intersection nodes per mm3 (120.3 × 103 ± 42.1 ×103), the average length of sinusoidal vessel per mm3 (5.4 × 103 ± 1.4 × 103 mm) and the percentage of vessel volume in tissue volume (15.3 ± 3.9) (means ± standard deviation). Several parameters of the BC network can be routinely extracted: the number of intersection node per mm3 (819.1 × 103 ± 180.7 ×103), the length of the BC network per mm3 (9.4 × 103 ± 0.7 × 103 mm) and the percentage of BC volume of the total volume (3.4 ± 0.005). The current protocols have several advantages: i) The quantitative parameters of hepatocytes and BC as well as sinusoidal networks can be extracted from the same tissue block; ii) Reconstructions and quantifications performed as described in the current protocols can be used for quantitative mathematical modelling of the underlying mechanisms; iii) Protocols are presented for rat, pig and human livers and iv) The technique can be applied also with conventional paraffin slices.
Full-text · Article · Jan 2015 · Zeitschrift für Gastroenterologie
[Show abstract][Hide abstract] ABSTRACT: Introduction: Bile duct ligation (BDL) is commonly used to study cholestasis and periportal fibrosis. Despite extensive studies, the spatial-temporal organization of the biliary tree in BDL remains unclear. Methods: To analyze and quantify the biliary tree after BDL, C57Bl/6N mice underwent BDL or sham operation and were observed for 6 hours to 28 days (n = 6/group). At predefined time-points (6h, 1 d, 3 d, 5 d, 7 d, 14 d and 28 d), blood and liver samples were collected for analysis of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) to assess hepatic parenchymal injury and alkaline phosphatase (AP) activities as well as bilirubin for biliary damage. Liver samples were subjected to routine histology and confocal microscopy. Confocal 3D-reconstructions were performed for periportal bile ducts and their links to lobular bile canaliculi. Immunostaining was performed combining antibodies directed against DPPIV/CD26 to visualize the bile canaliculi and the apical membrane of the bile ducts, anti-keratin19 (KRT19) to stain the bile duct epithelial cells, glutamine synthetase to identify the pericentral region of the lobules, anti-donkey mouse IgG which served as an immunoglobulin binds to the sinusoidal endothelial cells thereby visualizing sinusoids. Hepatocyte nuclei were counter stained by DAPI. Results: BDL caused damage to hepatic parenchyma and biliary epithelia cells, as indicted by a significant substantial elevation of transaminases and AP, which peaked on POD28. Histopathological examination of the liver revealed that the fraction of BrdU positive bile duct cells at day 3, 5 and 7 increased in BDL livers with a peak on POD3 compared to sham-operated livers. Morphometric analysis revealed that the total number of bile duct cells per periportal field significantly increased by days 7, 14 and 28 in BDL but peaked a little later. Confocal microscopy revealed that the periportal bile ducts showed extensive branching and increased diameter in BDL mice as compared to sham operated mice. The average numbers of Hering canals occurring per unit length of the bile duct were significantly decreased at days 14 and 28 after BDL. Conclusion: Bile duct ligation leads to biliary tree damage, thus initiating a regenerative response which is culminated in a maximal hepatobiliary cell proliferation 3 days post BDL. Increased hepatobiliary cell proliferation likely manifest as a general enlargement of the biliary tree through enhanced branching and dilation of the ducts.
Full-text · Article · Jan 2015 · Zeitschrift für Gastroenterologie
[Show abstract][Hide abstract] ABSTRACT: Transcriptomics is a powerful tool for high-throughput gene expression profiling. Transcriptome microarray experiments conducted with RNA isolated from hepatocytes after exposure to toxicants enable a deep insight into the molecular mechanisms of hepatotoxicity. This understanding, along with structure-activity relationships underlying hepatotoxicity, will provide a novel strategy to design cost-effective and safer therapeutics. Transcriptomics studies conducted with established hepatotoxic drugs in various in vitro and in vivo hepatotoxicity test systems have contributed to the elucidation of the mechanistic basis of liver insults, which were later on substantiated at the proteomics and metabolomics levels. The present chapter is focused on comprehensive transcriptomics of cultured primary hepatocytes treated with chemicals by applying Affymetrix microarray technology. It also describes the detailed protocol for culturing of hepatocytes, their exposure to toxicants as well as sample collection, including RNA isolation, RNA target preparation and finally the hybridization to gene chips for microarray expression analysis.