TY - JOUR A1 - Sbiera, Silviu A1 - Ronchi, Cristina L. A1 - Leich, Ellen A1 - Henzel, Katharina A1 - Rosenwald, Andreas A1 - Allolio, Bruno A1 - Fassnacht, Martin T1 - Single Nucleotide Polymorphism Array Profiling of Adrenocortical Tumors - Evidence for an Adenoma Carcinoma Sequence? JF - PLoS ONE N2 - Adrenocortical tumors consist of benign adenomas and highly malignant carcinomas with a still incompletely understood pathogenesis. A total of 46 adrenocortical tumors (24 adenomas and 22 carcinomas) were investigated aiming to identify novel genes involved in adrenocortical tumorigenesis. High-resolution single nucleotide polymorphism arrays (Affymetrix) were used to detect copy number alterations (CNAs) and copy neutral losses of heterozygosity (cnLOH). Genomic clustering showed good separation between adenomas and carcinomas, with best partition including only chromosome 5, which was highly amplified in 17/22 malignant tumors. The malignant tumors had more relevant genomic aberrations than benign tumors, such as a higher median number of recurrent CNA (2631 vs 94), CNAs >100 Kb (62.5 vs 7) and CN losses (72.5 vs 5.5), and a higher percentage of samples with cnLOH (91% vs 29%). Within the carcinoma cohort, a precise genetic pattern (i.e. large gains at chr 5, 7, 12, and 19, and losses at chr 1, 2, 13, 17, and 22) was associated with a better prognosis (overall survival: 72.2 vs 35.4 months, P=0.063). Interestingly, >70% of gains frequent in beningn were also present in malignant tumors. Notch signaling was the most frequently involved pathway in both tumor entities. Finally, a CN gain at imprinted “IGF2” locus chr 11p15.5 appeared to be an early alteration in a multi-step tumor progression, followed by the loss of one or two alleles, associated with increased IGF2 expression, only in carcinomas. Our study serves as database for the identification of genes and pathways, such as Notch signaling, which could be involved in the pathogenesis of adrenocortical tumors. Using these data, we postulate an adenoma-carcinoma sequence for these tumors. KW - adenomas KW - cancer diagnosis KW - cancer detection KW - carcinogenesis KW - carcinomas KW - chromosomes KW - genetic loci KW - malignant tumors KW - notch signaling Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-97218 ER - TY - JOUR A1 - Grimm, Martin A1 - Gasser, Martin A1 - Bueter, Marco A1 - Strehl, Johanna A1 - Wang, Johann A1 - Nichiporuk, Ekaterina A1 - Meyer, Detlef A1 - Germer, Christoph T. A1 - Waaga-Gasser, Ana M. A1 - Thalheimer, Andreas T1 - Evaluation of immunological escape mechanisms in a mouse model of colorectal liver metastases N2 - Background: The local and systemic activation and regulation of the immune system by malignant cells during carcinogenesis is highly complex with involvement of the innate and acquired immune system. Despite the fact that malignant cells do have antigenic properties their immunogenic effects are minor suggesting tumor induced mechanisms to circumvent cancer immunosurveillance. The aim of this study is the analysis of tumor immune escape mechanisms in a colorectal liver metastases mouse model at different points in time during tumor growth. Methods: CT26.WT murine colon carcinoma cells were injected intraportally in Balb/c mice after median laparotomy using a standardized injection technique. Metastatic tumor growth in the liver was examined by standard histological procedures at defined points in time during metastatic growth. Liver tissue with metastases was additionally analyzed for cytokines, T cell markers and Fas/Fas-L expression using immunohistochemistry, immunofluorescence and RT-PCR. Comparisons were performed by analysis of variance or paired and unpaired t test when appropriate. Results: Intraportal injection of colon carcinoma cells resulted in a gradual and time dependent metastatic growth. T cells of regulatory phenotype (CD4+CD25+Foxp3+) which might play a role in protumoral immune response were found to infiltrate peritumoral tissue increasingly during carcinogenesis. Expression of cytokines IL-10, TGF-b and TNF-a were increased during tumor growth whereas IFN-g showed a decrease of the expression from day 10 on following an initial increase. Moreover, liver metastases of murine colon carcinoma show an up-regulation of FAS-L on tumor cell surface with a decreased expression of FAS from day 10 on. CD8+ T cells express FAS and show an increased rate of apoptosis at perimetastatic location. Conclusions: This study describes cellular and macromolecular changes contributing to immunological escape mechanisms during metastatic growth in a colorectal liver metastases mouse model simulating the situation in human cancer. KW - Krebs KW - colon carcinoma cells KW - carcinogenesis KW - human cancer Y1 - 2010 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-67899 ER - TY - THES A1 - Rached, Eva Katharina T1 - Neue Ansätze zur Entwicklung von Alternativmethoden zur Prüfung auf chronische Nierentoxizität N2 - Die Niere ist eines der wichtigsten Zielorgane für Toxizität, allerdings stellt die frühzeitige Erkennung einer Nierenschädigung und/oder kanzerogenen Wirkung infolge einer wiederholten Exposition gegenüber toxischen Verbindungen ein großes Problem dar, da traditionelle Marker für Nierenfunktionsstörungen wenig empfindlich sind. Daher ist es notwendig, verbesserte Testmethoden (Alternativmethoden) zur Prüfung auf chronische Nierentoxizität zu entwickeln. Ziel dieser Arbeit war es daher, mögliche Alternativmethoden zur Prüfung auf Nephrotoxizität nach wiederholter Exposition zu untersuchen. Zum einen wurden dazu in einem in vivo-Modell für chronische Nierentoxizität neue Biomarker für Stress und Gewebeschädigung untersucht, deren erhöhte Genexpression in mehreren Modellen für akute Schädigung des Nierengewebes gezeigt wurde, einschließlich kidney injury molecule-1 (KIM-1), Lipocalin-2 (LCN2), Clusterin (CLU), Osteopontin (OPN), tissue inhibitor of metalloproteinases-1 (TIMP-1), Vimentin (VIM) und Hämoxygenase-1 (HO-1). Diese Marker wurden nachfolgend auch in einem zellkulturbasierten in vitro-Modell untersucht. Ein weiterer Teil der Arbeit befasste sich mit Veränderungen der Zellteilung als möglicher Marker für die Früherkennung kanzerogener Effekte. Das in vivo-Modell bestand in einer Studie in männlichen F344/N-Ratten, die 14, 28 oder 90 Tage oral mit 0, 21, 70 oder 210 µg/kg Körpergewicht (KG) Ochratoxin A (OTA) behandelt wurden. OTA ist ein Mykotoxin, das in Ratten bei wiederholter Gabe eine Nierenschädigung und Nierenkrebs verursacht. Die Analyse der mRNA-Expression der neuen Biomarker in Nierengewebe zeigte bei Tieren, die mit 70 oder 210 µg/kg KG behandelt wurden, eine frühzeitige, zeit- und dosisabhängige Induktion von KIM-1, LCN2, TIMP-1, OPN und CLU, die mit histopathologischen Veränderungen in Form von Zelldegeneration und Regeneration einherging und das Fortschreiten der Schädigung gut widerspiegelte. Auch die mRNA-Expression von HO 1 und VIM wurde durch OTA moduliert, allerdings war eine Erhöhung nicht zu allen Zeitpunkten zu messen bzw. trat nicht so früh auf wie bei den anderen Markern. Effekte auf traditionelle Marker für Nephrotoxizität (Serum-Kreatinin, N-Acetyl-β-D-glucosaminidase und γ-Glutamyltransferase im Urin) wurden im Vergleich zu den neuen Markern zu einem späteren Zeitpunkt und zumeist nur in der Hochdosisgruppe festgestellt. Zusätzlich zu den Effekten auf die Genexpression konnte in den Zielzellen von OTA im proximalen Tubulusepithel eine erhöhte Proteinexpression von KIM-1, CLU, OPN und VIM gezeigt werden; nur für KIM-1 wurde allerdings auch im Urin eine erhöhte Konzentration nachgewiesen, die mit den Effekten auf die mRNA- und Proteinkonzentration im Gewebe korrelierte. Damit stellt KIM-1 in dieser Studie hinsichtlich Empfindlichkeit und Messbarkeit den empfindlichsten Biomarker für Nephrotoxizität dar. Die Untersuchung der Zellteilung nach wiederholter Gabe von OTA zeigte einen dramatischen, zeit- und dosisabhängigen Anstieg der Proliferation von proximalen Tubulusepithelzellen in Nieren von Tieren, die mit 70 oder 210 µg/kg KG behandelt wurden. Dagegen wurden nach wiederholter Exposition gegenüber 21 µg/kg KG über 90 Tage keine OTA-abhängigen Effekte auf die renale Zellproliferation festgestellt. Somit korrelieren die Veränderungen der Zellteilung in der Niere in der 90-Tages-Studie sehr gut mit dem Ergebnis der 2-Jahres-Kanzerogenitätsstudie mit OTA, in der Nierentumoren nur nach Behandlung mit 70 oder 210 µg/kg KG auftraten. Ausgehend von den verschiedenen Endpunkten für Toxizität, die in der Studie untersucht wurden, liegt der no-observed-adverse-effect-level (NOAEL) bei 21 µg/kg KG OTA. Dies entspricht dem NOAEL der 2-Jahres-Kanzerogenitätsstudie. In einem weiteren Teil der Arbeit wurden die neuen in vivo-Biomarker für Nephrotoxizität in NRK 52E-Zellen als in vitro-Modell ausgetestet. Allerdings konnte eine erhöhte mRNA-Expression von KIM-1, einem sensitiven Marker in vivo, nach 24 oder 48 Stunden Behandlung mit verschiedenen nephrotoxischen Modellverbindungen (OTA, Kaliumbromat (KBrO3), Cisplatin oder Cadmiumchlorid (CdCl2)) in den Zellen nicht nachgewiesen werden. Die mRNA-Expression anderer Marker (VIM, CLU, TIMP-1, LCN2, OPN) war dagegen in unbehandelten Zellen bereits so hoch, dass die Behandlung mit Nephrotoxinen zu keiner weiteren Induktion führte. Allein die Gen- und Proteinexpression von HO-1 wurde durch CdCl2, KBrO3 und OTA induziert und könnte daher einen potentiellen Marker für screening-Studien in vitro darstellen. Insgesamt war der Nachweis zytotoxischer Wirkungen jedoch der empfindlichste Endpunkt in der Zellkultur. Die Ergebnisse stützen somit die Verwendung der neuen in vivo-Biomarker als gewebespezifische Marker für Nephrotoxizität in vitro nicht. N2 - The kidney is a main target organ of toxicity, but early detection of kidney damage and/or carcinogenic effects following the repeated exposure to toxic substances presents a major problem, since traditional markers of renal malfunction suffer from lack of sensitivity. Therefore, nephrotoxic effects are often detected only in long-term experiments in animals. Ethical reasons as well as the immense time and costs required for these animal studies have prompted the search for alternative methods by which animal numbers and duration of studies can be reduced. A further, albeit challenging, attempt is to replace experiments in animals by studies in vitro. The aim of this work was to test possible alternative methods for the detection of nephrotoxicity after repeated exposure. One the one hand, the expression of new biomarkers of stress and tissue damage was studied in an in vivo-model of chronic nephrotoxicity; enhanced gene expression of these biomarkers, including kidney injury molecule-1 (KIM-1), lipocalin-2 (LCN2), clusterin (CLU), osteopontin (OPN), tissue inhibitor of metalloproteinases-1 (TIMP-1), vimentin (VIM), and heme oxygenase-1 (HO-1), had been demonstrated before in several models of acute kidney damage. In addition to the experiments in vivo, the markers were also studied in a cell culture-based in vitro-model to assess their use as sensitive endpoints of toxicity in vitro. A further part of this work included the determination of cell proliferation as potential early marker of toxin-induced carcinogenic effects. As an in vivo-model, a toxicity study in male F344/N rats was performed. Rats were treated 14, 28 or 90 days with 0, 21, 70 or 210 µg/kg body weight (bw) ochratoxin A (OTA) by oral administration. OTA is a mycotoxin that is known to cause kidney damage and renal tumors in rats after repeated exposure. Analysis of mRNA expression of the new biomarkers showed early, time- and dose-dependent induction of KIM-1, LCN2, TIMP-1, OPN and CLU in kidney tissue of animals treated with 70 or 210 µg/kg bw. The induction of these biomarkers accompanied histopathological changes like cell degeneration and regeneration and mirrored well the progression of tissue damage. mRNA expression of HO-1 and VIM was also modulated by OTA, but overexpression was not evident at all time points or occurred later than for the other markers. Compared with the new biomarkers, effects on traditional markers of nephrotoxicity (serum creatinine, urinary N-acetyl-β-D-glucosaminidase and γ-glutamyltransferase) were restricted to later time points and the high dose group. In addition to the effects on gene expression, enhanced protein expression of KIM-1, CLU, OPN and VIM was observed in target cells of OTA in the proximal tubule epithelium; however, only for KIM-1, increased protein levels were also measured in urine, which correlated with the effects on the gene and protein expression in kidney tissue. Therefore, KIM-1 appeared to be the most sensitive biomarker of nephrotoxicity in this study. The study of the renal cell division after repeated administration of OTA demonstrated a dramatic, time- and dose-dependent increase in the proliferation of proximal tubule epithelial cells in kidneys of rats exposed to 70 or 210 µg/kg bw. In contrast, no OTA-dependent effects on renal cell proliferation were observed after repeated administration of 21 µg/kg bw. Thus, changes of renal cell proliferation in this 90-day-study correlate well with the results of the 2-year-carcinogenicity study with OTA, where renal tumors were only detected at 70 or 210 µg/kg bw. Based upon the different endpoints of toxicity determined in this study, the no-observed-adverse-effect-level (NOAEL) is 21 µg/kg KG OTA. This is consistent with the result of the 2-year-carcinogenicity study. In another part of this work, the new in vivo biomarkers of nephrotoxicity were studied in NRK-52E cells as in vitro-model. However, mRNA expression of KIM-1, one of the best biomarkers in vivo, was not detected in the cells after treatment for 24 or 48 hours with several nephrotoxic model substances (OTA, potassium bromate (KBrO3), cisplatin or cadmium chloride (CdCl2)). In contrast, high basal mRNA expression of other markers (VIM, CLU, TIMP-1, LCN2, OPN) was evident even in untreated cells and treatment with nephrotoxins did not further enhance marker gene expression. Only in the case of HO-1, both gene and protein expression were induced by CdCl2, KBrO3 and OTA, and could therefore represent potential markers in screening studies in vitro. In summary, measurement of cytotoxicity was still the most sensitive endpoint of toxicity in vitro. Thus, results from this study do not support the use of the new in vivo-biomarkers as tissue-specific markers of nephrotoxicity in vitro. KW - Niere KW - Toxizität KW - Biomarker KW - Carcinogenese KW - Niere KW - Toxizität KW - Biomarker KW - Carcinogenese KW - nephrotoxicity KW - biomarker KW - carcinogenesis Y1 - 2009 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-47812 ER -