Markus Schug

Role of thioredoxin reductase 1 and thioredoxin interacting protein in prognosis of breast cancer

IntroductionThe purpose of this work was to study the prognostic influence in breast cancer of thioredoxin reductase 1 (TXNRD1) and thioredoxin interacting protein (TXNIP), key players in oxidative stress control that are currently evaluated as possible therapeutic targets.MethodsAnalysis of the association of TXNRD1 and TXNIP RNA expression with the metastasis-free interval (MFI) was performed in 788 patients with node-negative breast cancer, consisting of three individual cohorts (Mainz, Rotterdam and Transbig). Correlation with metagenes and conventional clinical parameters (age, pT stage, grading, hormone and ERBB2 status) was explored. MCF-7 cells with a doxycycline-inducible expression of an oncogenic ERBB2 were used to investigate the influence of ERBB2 on TXNRD1 and TXNIP transcription.ResultsTXNRD1 was associated with worse MFI in the combined cohort (hazard ratio = 1.955; P < 0.001) as well as in all three individual cohorts. In contrast, TXNIP was associated with better prognosis (hazard ratio = 0.642; P < 0.001) and similar results were obtained in all three subcohorts. Interestingly, patients with ERBB2-status-positive tumors expressed higher levels of TXNRD1. Induction of ERBB2 in MCF-7 cells caused not only an immediate increase in TXNRD1 but also a strong decrease in TXNIP. A subsequent upregulation of TXNIP as cells undergo senescence was accompanied by a strong increase in levels of reactive oxygen species.ConclusionsTXNRD1 and TXNIP are associated with prognosis in breast cancer, and ERBB2 seems to be one of the factors shifting balances of both factors of the redox control system in a prognostic unfavorable manner.

Tracking of human cells in mice.

Tracking and tracing of transplanted cells in mice is required in many fields of research. Examples are transplantation of stem cells into organs of mice to study their differentiation capacity and injection of tumor cells to examine metastatic behavior. In the present study we tested the lipid dye CM-DiI and red fluorescent nanoparticles Qdot655 for their applicability in tagging and tracing of human cells in mice. Labeling of different cell types, including MCF-7 human breast cancer cells, human cord blood derived cells, human NeoHep cells and human hepatopancreatic precursor cells, is technically easy and did not compromise further cell culture. After transplantation of CM-DiI or Qdot655 marked cells, red fluorescent structures could be detected already in unprocessed paraffin slices of the studied organs, namely liver, lung, pancreas, kidney, spleen and bone marrow. Next, we examined whether the red fluorescent structures represent the transplanted human cells. For this purpose, we established an in situ hybridization (ISH) technique that allows clear-cut differentiation between human and murine nuclei, based on simultaneous hybridization with human alu and mouse major satellite (mms) probes. We observed a high degree of coincidence between CM-DiI-marked cells and alu positive nuclei. However, also some mms positive cells contained CM-DiI, suggesting phagocytosis of the transplanted CM-DiI-marked cells. The degree of such CM-DiI-positive mouse cells depended on the cell type and route of administration. From a technical point of view it was important that CM-DiI-positive structures in paraffin slices remained fluorescent also after ISH. In contrast, Qdot655 positive structures faded during further staining procedures. In conclusion, marking of cells with CM-DiI or Qdot655 prior to transplantation facilitates recovery of human cells, since a high fraction of positive structures in the host’s tissue originate from the transplanted cells. However, CM-DiI or Qdot655 positive staining of individual cells in transplanted tissues is not sufficient to prove their human origin. Additional procedures, such as ISH with alu-probes, are essential, when characterizing individual cells.

Dexamethasone-dependent versus -independent markers of epithelial to mesenchymal transition in primary hepatocytes

Abstract Recently, epithelial to mesenchymal transition (EMT) has been shown to represent a feature of dedifferentiating hepatocytes in vitro. Three-dimensional soft collagen gels can antagonize but not completely abolish this effect. Hormonal additives to culture media are known to maintain differentiated hepatocyte functions. Therefore, we studied whether insulin and dexamethasone antagonize EMT in cultured hepatocytes. Both hormones antagonized but not completely abolished certain morphological features of EMT. Dexamethasone antagonized acquisition of fibroblastoid shape, whereas insulin favored bile canaliculi formation. In a subsequent step, we analyzed expression of a battery of EMT-related genes. Of all markers tested, vimentin and snail-1 correlated best with morphological features of EMT. Interestingly, dexamethasone reduced expression levels of both vimentin and snail-1, whereas the influence of insulin was less pronounced. An important result of this study is that 12 out of 17 analyzed EMT markers were transcriptionally influenced by dexamethasone (vimentin, snail-1, snail-2, HNF4α, Twist-1, ZEB2, fibronectin, occludin, MMP14, claudin-1, cytokeratin-8, and cytokeratin-18), whereas the remaining factors seemed to be less dependent on dexamethasone. In conclusion, EMT markers in hepatocytes can be classified as dexamethasone-dependent versus -independent.

A physiologically based toxicokinetic modelling approach to predict relevant concentrations for in vitro testing

Our study was performed in the context of an in vitro primary hepatic cell culture as an alternative for the in vivo cancerogenic bioassay. The 29 substances which are to be used in the in vitro primary hepatic cell culture have been tested in 2-year bioassays and a 14-day short term study. The aim of this modelling study was to simulate the concentration–time profile of the compounds when given by the oral route at the doses tested in the previous studies taking into account the percentage of the dose absorbed. The model contained seven tissue compartments with uptake from the gastrointestinal tract into the portal vein. Because the primary hepatic cell culture is metabolically competent and the primary interest was to model the concentration in the portal vein, the hepatic vein and the systemic circulation (blood) in the beginning we did not include elimination. Partitioning between blood and tissues was calculated according to a published biologically based algorithm. The substances’ kinetic profile differed according to their blood: tissue partitioning. Maximal concentrations in portal vein, hepatic vein and the blood depended mainly on the dose and the fraction absorbed which were the most critical parameters in this respect. Our study demonstrates an application of BPTK modelling for the purpose to simulate concentrations for planning the doses for an in vitro study. BPTK modelling seems to be a better approach than using data from in vitro studies on cytotoxicity.

Reversible Manipulation of Apoptosis Sensitivity in Cultured Hepatocytes by Matrix-Mediated Manipulation of Signaling Activities

Hepatocytes in culture are a valuable tool to investigate mechanisms involved in the response of the liver to cytokines. However, it is well established that hepatocytes cultured as monolayers on dried stiff collagen dedifferentiate, loosing specialized liver functions. In contrast, softer matrix systems like gelled collagen help to preserve these structural and functional features. We show that the de-differentiation process induced in conventional dry collagen is a reversible consequence of a specific signaling network constellation triggered by the extracellular matrix that results in apoptosis resistance. A dried stiff collagen activates Akt and ERK1/2 pathways that results in apoptosis resistance. In contrast to stiff collagen, a soft collagen gel does not activate these pathways keeping the hepatocytes in a state where they remain sensitive to TGF-beta-induced apoptosis. Finally, we show that matrix-induced apoptosis resistance is reversible by re-plating cells from dried stiff to soft gel collagen. Practical consequences of these observations are that differentiated functions of hepatocytes, such as metabolism, endocytosis, and apoptosis, should be studied in hepatocyte sandwiches. On the other hand, proliferation and regeneration associated signaling can better be studied in hepatocytes cultured on collagen monolayers. In this chapter we focus on mechanisms that influence apoptosis sensitivity in cultured mouse hepatocytes.