Thilo Spruss

Transport of lipids from golgi to plasma membrane is defective in tangier disease patients and Abc1-deficient mice.

Mutations in the gene encoding ATP-binding cassette transporter 1 ( ABC1) have been reported in Tangier disease (TD), an autosomal recessive disorder that is characterized by almost complete absence of plasma high-density lipoprotein (HDL), deposition of cholesteryl esters in the reticulo-endothelial system (RES) and aberrant cellular lipid trafficking. We demonstrate here that mice with a targeted inactivation of Abc1 display morphologic abnormalities and perturbations in their lipoprotein metabolism concordant with TD. ABC1 is expressed on the plasma membrane and the Golgi complex, mediates apo-AI associated export of cholesterol and phospholipids from the cell, and is regulated by cholesterol flux. Structural and functional abnormalities in caveolar processing and the trans-Golgi secretory pathway of cells lacking functional ABC1 indicate that lipid export processes involving vesicular budding between the Golgi and the plasma membrane are severely disturbed.

Activated hepatic stellate cells promote tumorigenicity of hepatocellular carcinoma

Liver cirrhosis is the main risk factor for the development of hepatocellular carcinoma (HCC). Activated hepatic stellate cells (HSC) are the effector cells of hepatic fibrosis and also infiltrate the HCC stroma where they might play a critical role in HCC progression. Here we aimed to analyze the effects of activated HSC on the proliferation and growth of HCC cell lines in vitro and in vivo. Conditioned media (CM) collected from HSC significantly induced proliferation and migration of HCC cells cultured in monolayers. In a 3‐dimensional spheroid coculture system, HSC promoted HCC growth and diminished the extent of central necrosis. In accordance, in vivo simultaneous implantation of HSC and HCC cells into nude mice promoted tumor growth and invasiveness, and inhibited necrosis formation. As potential mechanism of the tumorigenic effects of HSC we identified activation of NFkappaB and extracellular‐regulated kinase (ERK) in HCC cells, two signaling cascades that play a crucial role in HCC progression. In summary, our data indicate that stromal HSC promotes HCC progression and suggest the HSC–HCC interaction as an interesting tumor differentiation‐independent target for therapy of this highly aggressive cancer. (Cancer Sci 2009; 100: 646–653)

Standardized kinetic microassay to quantify differential chemosensitivity on the basis of proliferative activity

SummaryConventionally in vitro cytotoxicity assays are performed as single-end-point determinations. To compensate for the diversity of growth rates among different cell lines in this report we describe a computerized kinetic chemosensitivity assay based on quantification of biomass by staining cells with crystal violet. As a prerequisite four human breast cancer cell line (MDA-MB-231, MCF-7, T-47-D and ZR-75-1) were characterized with regard to oestrogen and progesterone receptor content, modal chromosome number and proliferation kinetics depending on the number of passages in culture. With prolonged time in culture for ZR-75-1 exposed to various concentrations of cisplatinum a dose-related increase in drug effect was observed. Owing to a correction of the T/C values for the initial cell mass (at the time when drug is added) a sharp distinction between cytostatic and cytocidal drug effects becomes obvious in plots of corrected T/C values versus time of incubation. The influence of the untreated control on the corrected T/C values and possible time courses of theoretical inhibition profiles (reflecting cytostatic, transient cytotoxic or cytocidal drug effects as well as development of resistance) and their relationship to the corresponding growth curves of drugtreated cells are discussed. Chemosensitivity assays with diethylstilbestrol dipropionate, tamoxifen, melphalan, cisplatinum, vinblastine, Adriamycin and 5-fluorouracil prove the theoretical considerations to be true for MDA-MB-231, MCF-7, T-47-D and ZR-75-1 human breast cancer cell lines in practice.

Down-regulation of CYLD expression by Snail promotes tumor progression in malignant melanoma

High malignancy and early metastasis are hallmarks of melanoma. Here, we report that the transcription factor Snail1 inhibits expression of the tumor suppressor CYLD in melanoma. As a direct consequence of CYLD repression, the protooncogene BCL-3 translocates into the nucleus and activates Cyclin D1 and N-cadherin promoters, resulting in proliferation and invasion of melanoma cells. Rescue of CYLD expression in melanoma cells reduced proliferation and invasion in vitro and tumor growth and metastasis in vivo. Analysis of a tissue microarray with primary melanomas from patients revealed an inverse correlation of Snail1 induction and loss of CYLD expression. Importantly, tumor thickness and progression-free and overall survival inversely correlated with CYLD expression. Our data suggest that Snail1-mediated suppression of CYLD plays a key role in melanoma malignancy.

Functional implication of BMP4 expression on angiogenesis in malignant melanoma

Analyses of malignant melanomas revealed a strong expression of bone morphogenic proteins (BMPs) and their autocrine effect in promoting cell invasion and migration. Here, we report a paracrine effect of BMPs on the vascular network. Both BMP2 and BMP4 induced tube formation as well as the migratory efficiency of microvascular endothelial cells. Melanoma cells with reduced BMP activity attracted less endothelial cells in invasion assays than control cells. Furthermore, reduction of BMPs in melanoma cells had a strong effect on vasculogenic mimicry. Tube formation on matrigel was analysed for melanoma cells as well as in co-cultures of endothelial and melanoma cells. Melanoma cells with reduced BMP activity were not capable of forming cord-like structures by themselves and additionally inhibited tube formation of the endothelial cells. Genes involved in angiogenesis turned out to be strongly downregulated in these cell clones. Tumors derived from cells with impaired BMP activity showed reduced tumor growth or large necrotic areas owing to lack of angiogenesis in in vivo analyses.

External Carotid Artery Territory Ischemia Impairs Outcome in the Endovascular Filament Model of Middle Cerebral Artery Occlusion in Rats

BACKGROUND AND PURPOSE Middle cerebral artery occlusion (MCAO) by an intraluminal filament is a widely accepted animal model of focal cerebral ischemia. In this procedure, cutting of the external carotid artery (ECA) is a prerequisite for thread insertion. However, the implications of ECA transsection have not yet been described. METHODS After 90 minutes of filament MCAO or sham surgery, rats were evaluated for up to 14 days in terms of body weight development, core temperature, and motor performance. Repeated in vivo MRI of the head and neck was performed for quantification of brain edema and infarct volume. The temporal muscles were histologically analyzed postmortem. RESULTS In 47% of all rats, ischemic tissue damage to the ipsilateral ECA area, including temporal, lingual, and pharyngeal musculature, was detectable by MRI. Histology of temporal muscles confirmed acute ischemic myopathy. Animals with ECA territory ischemia (ECA-I) showed delayed body weight development and poorer recovery of motor function. There was no difference in the extent of brain edema or final cerebral lesion size between ECA-I-affected and unaffected rats. CONCLUSIONS Filament MCAO was complicated by the consequences of ECA ischemia in approximately half of all rats. Impaired mastication and swallowing functions restricted ingestion and resulted in postsurgical body weight loss and worse motor performance. Impaired cerebral microperfusion resulting from dehydration and reduced spontaneous motor activity resulting from reduced food and water uptake might have contributed to poorer neurological recovery in ECA ischemic rats. Thus, adverse effects caused by extracerebral ischemia with potential impact on outcome have to be considered in this stroke model.

“Hit-and-Run” Transformation by Adenovirus Oncogenes

ABSTRACT According to classical concepts of viral oncogenesis, the persistence of virus-specific oncogenes is required to maintain the transformed cellular phenotype. In contrast, the “hit-and-run” hypothesis claims that viruses can mediate cellular transformation through an initial “hit,” while maintenance of the transformed state is compatible with the loss (“run”) of viral molecules. It is well established that the adenovirus E1A and E1B gene products can cooperatively transform primary human and rodent cells to a tumorigenic phenotype and that these cells permanently express the viral oncogenes. Additionally, recent studies have shown that the adenovirus E4 region encodes two novel oncoproteins, the products of E4orf6 and E4orf3, which cooperate with the viral E1A proteins to transform primary rat cells in an E1B-like fashion. Unexpectedly, however, cells transformed by E1A and either E4orf6 or E4orf3 fail to express the viral E4 gene products, and only a subset contain E1A proteins. In fact, the majority of these cells lack E4- and E1A-specific DNA sequences, indicating that transformation occurred through a hit-and-run mechanism. We provide evidence that the unusual transforming activities of the adenoviral oncoproteins may be due to their mutagenic potential. Our results strongly support the possibility that even tumors that lack any detectable virus-specific molecules can be of viral origin, which could have a significant impact on the use of adenoviral vectors for gene therapy.

CD137 is expressed on blood vessel walls at sites of inflammation and enhances monocyte migratory activity

The cytokine receptor CD137 is a member of the TNF receptor family and a potent T cell costimulatory molecule. Its ligand is expressed on antigen presenting cells as a transmembrane protein and it too can deliver signals into the cells it is expressed on (reverse signaling). In monocytes, immobilized CD137 protein induces activation, prolongation of survival and proliferation. Here we show that recombinant immobilized CD137 protein enhances migration of monocytes in vitro. Further, CD137 expression on spheroids leads to a significantly enhanced infiltration by monocytes. The migration‐inducing activity of CD137 could be confirmed in vivo. Matrigel, which was coated with recombinant CD137 protein and was inserted into the flanks of mice attracted large numbers of monocytes and was heavily infiltrated by these cells. In vivo, expression of CD137 by blood vessel walls at sites of inflammation was detectable by immunohistochemistry. CD137 expression is inducible by proinflammatory cytokines in endothelial cells, suggesting that a physiological function of CD137 may be the facilitation of monocyte extravasation in inflammatory tissues.—Drenkard D., Becke F. M., Langstein J., Spruss T., Kunz‐Schughart L.A., Tan T.E., Lim Y.C., Schwarz H. CD137 is expressed on blood vessel walls at sites of inflammation and enhances monocyte migratory activity. FASEB J. 21, 456–463 (2007)

Quantitation of hyaluronidases by the Morgan-Elson reaction : comparison of the enzyme activities in the plasma of tumor patients and healthy volunteers

The Morgan-Elson reaction, a method for the determination of hyaluronidase activity, was optimized for the quantitation of the enzyme in biological material. Based on HPLC and spectrometric (UV-Vis, LC-MS) studies, the structure of the red-colored product (mesomeric forms of N3-protonated 3-acetylimino-2-(4-dimethylaminophenyl)methylidene-5-(1,2-++ +dihydroxyethyl)furane) formed by condensation of chromogen III with p-dimethylaminobenzaldehyde is proposed. Activities corresponding to > or = 0.1 IU of endogenous and therapeutically administered hyaluronidase can be detected in 50 microl samples. Application of the method for the determination of the enzyme in plasma of tumor patients revealed no difference in activity levels, interindividual variability and pH profile compared to healthy volunteers.

The novel gene MIA2 acts as a tumour suppressor in hepatocellular carcinoma

Background: Melanoma inhibitory activity 2 (MIA2) is a novel gene of the MIA gene family. The selective expression of MIA2 in hepatocytes is controlled by hepatocyte nuclear factor (HNF) 1 binding sites in the MIA2 promotor. In contrast, in most hepatocellular carcinomas (HCC) MIA2 expression is down-regulated or lost. Aim: In this study we examined the regulation and functional role of MIA2 in hepatocancerogenesis. Methods and results: In HCC cell lines and tissues HNF-1 expression was lower than in primary human hepatocytes (PHH) and corresponding non-tumorous tissue, respectively, and correlated significantly with the down-regulation of MIA2 expression. Re-expression of HNF-1 in HCC cells reinduced MIA2 in HCC cells to similar levels as found in PHH. Further, MIA2 was re-expressed in HCC cell lines by stable transfection, and the generated cell clones revealed a strongly reduced invasive potential and proliferation rate in vitro. In line with these findings treatment of HCC cells with recombinant MIA2 inhibited proliferation and invasion. In nude mice MIA2 re-expressing HCC cells grew significantly slower and revealed a less invasive growth pattern. Immunohistochemical analysis of a tissue microarray containing HCC and corresponding non-cancerous liver tissue of 85 patients confirmed reduced MIA2 expression in HCC. Furthermore, MIA2 negative HCC tissue showed a significantly higher Ki67 labelling index and loss of MIA2 expression correlated significantly with more advanced tumour stages. Conclusion: This study presents MIA2 as an inhibitor of HCC growth and invasion both in vitro and in vivo, and consequently, as a tumour suppressor of HCC. Further, our findings indicate a novel mechanism, how loss of HNF-1 expression in HCC affects tumorigenicity via down-regulation of MIA2.