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The covalent binding of [3H]aflatoxin B1 (AF) to liver DNA was determined, 6 h after oral administration to male rabbits. A Covalent Binding Index, CBI (flmol AF/mol DNA-P)/(mmol AF/kg b. w.) = 8,500 was found. Pretreatment of rabbits with AF coupled to bovine serum albumin in Freund's adjuvant led to the production of AF-directed antibodies. Administration of [3H]AF to such immunized rabbits resulted in a CJH of only 2,500, i.e., the iiDJ{.lUnization provided a protection by a factor of more than 3. Although this is encouraging evidence for the potential of active immunization against genotoxic carcinogens, a nurober of pointswill have to be clarified, such as the time course for the DNA binding and the question of a possible shift to other target cells.
Inhaltsübersicht zum Schwerpunktthema: - Erkennung, Therapie und Nachsorge von Tumoren - Tumorkonforme Strahlentherapie - Kinder mit Hirntumoren - Autologe Transplantation von Blutstammzellen - Tumorimmunität am Beispiel des malignen Melanoms - Immuntherapie: ein Antikörper gegen Magenkrebs - Brustkrebs - die Lymphknoten und der Einfluss der Gene - Krebs in der unteren Speiseröhre u.a.
Inhaltsübersicht zum Schwerpunktthema: - Krebsentstehung auf der Ebene der Moleküle - Veränderungen am Erbgut ohne direkte DNA-Schädigung - Magenlymphom: von der Infektion zum Tumor - Chaos in der Erbsubstanz - Hautkrebs bei Fischen: ein Tumorgen und die Folgen - Ewiges Leben - ein Albtraum? - Wilms-Tumoren - komplexer als vermutet u.a.
The first goal of this study was to develop cell lines with a stable expression of bio-fluorescent topo II and topo I. This was successfully achieved using a bicistronic vector system. Control experiments showed that proteins of expected size were expressed, and that GFP-tagged topos I, IIa, and IIb were active in the cells and fully integrated in the endogenous pools of the enzymes. These cell-lines provided a novel tool for investigating the cell biology of human DNA topoisomerases. Our most important finding was, that both types of mammalian topoisomerases are entirely mobile proteins that are in continuous and rapid flux between all compartments of the nucleus and between the cytososl and the chromosomes of mitotic cells. This was particularly surprising with regard to topo II, which is considered to be a structural component of the nuclear matrix and the chromosome scaffold. We must conclude that if this was the case, then these architectural structures appear to be much more dynamic than believed until now. In this context it should also be mentioned, that the alignment of topo II with the central axes of the chromosome arms, which has until now been considered a hall-mark of the enzyme’s association with the chromosomal scaffold, is not seen in vivo and can be demonstrated to be to some extent an artefact of immunohistochemistry. Furthermore, we show that the two isoforms of topo II (a and b) have a different localisation during mitotic cell division, supporting the general concept that topo II functions at mitosis are exclusively assigned to the a-form, whereas at interphase the two isoenzymes work in concert. Despite unrestricted mobility within the entire nuclear space, topoisomerases I and II impose as mostly nucleolar proteins. We show that this is due to the fact that in the nucleoli they are moving slower than in the nucleoplasm. The decreased nucleolar mobility cannot be due to DNA-interactions, because compounds that fix topoisomerases to the DNA deplete them from the nucleoli. Interestingly, the subnucleolar distribution of topoisomerases I and II was complementary. The type II enzyme filled the entire nucleolar space, but excluded the fibrial centers, whereas topo I accumulated at the fibrial centers, an allocation directed by the enzyme’s N-terminus. During mitosis, it also mediates association with the nucleolar organising regions of the acrocentric chromosomes. Thus, topo I stays associated with the rDNA during the entire cell-cycle and consistently colocalizes there with RNA-polymerase I. Finally, we show that certain cancer drugs believed to act by stabilising covalent catalytic DNA-intermediates of topoisomerases, do indeed immobilize the enzymes in living cells. Interestingly, these drugs do not target topoisomerases in the nucleoli but only in the nucleoplasm.
In a variety of established tumour cell lines, but also in primary mammary epithelial cells metalloprotease-dependent transactivation of the EGFR, and EGFR characteristic downstream signalling events were observed in response to stimulation with physiological concentrations of GPCR agonists such as the mitogens LPA and S1P as well as therapeutically relevant concentrations of cannabinoids. Moreover, this study reveals ADAM17 and HB-EGF as the main effectors of this mechanism in most of the cancer cell lines investigated. However, depending on the cellular context and GPCR agonist, various different members of the ADAM family are selectively recruited for specific ectodomain shedding of proAR and/or proHB-EGF and subsequent EGFR activation. Furthermore, biological responses induced by LPA or S1P such as migration in breast cancer and HNSCC cells, depend on ADAM17 and proHB-EGF/proAR function, respectively, suggesting that highly abundant GPCR ligands may play a role in tumour development and progression. Moreover, EGFR signal transactivation could be identified as the mechanistic link between cannabinoid receptors and the activation of mitogen activated protein kinases (MAPK) ERK1/2 as well as pro-survival Akt/PKB signalling. Depending on the cellular context, cannabinoid-induced signal cross-communication was mediated by shedding of proAmphiregulin and/or proHB-EGF by ADAM17. Most importantly, our data show that concentrations of THC comparable to those detected in the serum of patients after THC administration accelerate proliferation of cancer cells instead of apoptosis and thereby may contribute to cancer progression in patients.
The Ras/RAF/MEK/ERK cascade is a central cellular signal transduction pathway involved in cell proliferation, differentiation, and survival where RAF kinases are pivotal kinases implicated in cancer. The development of specific irreversible kinase inhibitors is a rewarding but difficult aim. CI-1033 was developed to irreversibly inhibit erbB receptor tyrosine kinases by reacting to the Cys113 residue (p38alpha MAP kinase numbering) of the kinase domain. In this study we tried a similar approach to target the RAF oncoproteins which posses a similar cysteine at position 108 in the hinge region between the small n-lobe and the large c-lobe of the kinase domain. A novel synthetic approach including a lyophilization step allowed us the synthesis of a diphenyl urea compound with an epoxide moiety (compound 1). Compound 1 possessed inhibitory activity in vitro. However our time kinetics experiments and mass spectroscopic studies clearly indicate that compound 1 does not react covalently with the cysteine residue in the hinge region. Moreover, in cell culture experiments, a strong activation of the RAF signaling pathway was observed, an effect which is known from several other RAF kinase inhibitors and is here reported for the first time for a diphenyl urea compound, to which the clinically used unspecific kinase inhibitor BAY 43-9006 (Sorafinib, Nexavar) belongs. Although activation was apparently independent on B- and C-RAF hetero-oligomerization in vitro, in vivo experiments support such a mechanism as the activation did not occur in starved knockout cells lacking either B-RAF or C-RAF. Furthermore, we developed a mathematical model of the Ras/RAF/MEK/ERK cascade demonstrating how stimuli induce different signal patterns and thereby different cellular responses, depending on cell type and the ratio between B-RAF and C-RAF. Based on biochemical data for activation and dephosphorylation, we set up differential equations for a dynamical model of the Ras/RAF/MEK/ERK cascade. We find a different signaling pattern and response result for B-RAF (strong activation, sustained signal) and C-RAF (steep activation, transient signal). We further support the significance of such differential modulatory signaling by showing different RAF isoform expression in various cell lines and experimental testing of the predicted kinase activities in B-RAF, C-RAF as well as mutated versions. Additionally the effect of the tumor suppressor DiRas3 (also known as Noey2 or ARHI) on RAF signaling was studied. I could show that DiRas3 down-regulates the mitogenic pathway by inhibition of MEK, a basis for a refined model of the Ras/RAF/MEK/ERK cascade.
Das follikuläre Lymphom ist eines der häufigsten Non-Hodgkin Lymphome und überwiegend eine Erkrankung des erwachsenen Menschen. In der WHO-Klassifikation ist es als ein Lymphom von Keimzentrumszellen definiert, das follikulär und/oder diffus wachsen kann. Zur Subklassifikation follikulärer Lymphome empfiehlt die WHO-Klassifikation eine Unterscheidung der Grade 1, 2 und 3 durch Auszählen der Zentroblasten pro zehn Gesichtsfelder in starker Vergrößerung. Beim Grad 3A liegen neben Zentroblasten auch Zentrozyten vor. FL Grad 3B bestehen ausschließlich aus Zentroblasten. Hinsichtlich der Zytomorphologie, Immunhistologie und Genetik bestehen deutliche Unterschiede zwischen FL Grad 1, 2 und 3A gegenüber FL Grad 3B. In der vorliegenden Arbeit konnte gezeigt werden, dass die weit überwiegende Zahl der follikulären Lymphome Grad 1, 2 und 3A ein prädominant follikuläres Wachstumsmuster aufwies. Ein follikulärer und diffuser Wuchstyp lag seltener vor. Noch seltener war ein überwiegend bzw. „rein“ diffuses Wachstumsmuster. Die mitotische und proliferative Aktivität stieg mit dem Tumorgrad linear an. Hinsichtlich der CD10 Reaktivität, der BCL-2 und p53 Expression sowie des Nachweises einer sekretorischen Differenzierung ergaben sich beim Vergleich der FL Grad 1 bis 3A keine statistisch signifikanten Unterschiede. Die BCL-2 Expression nahm allerdings bei den FL1-3A mit zunehmendem Grad ab. In zytogenetischen Untersuchungen wurden in allen follikulären Lymphomen Grad 1 bis 3A primäre bzw. sekundäre Chromosomenaberrationen gefunden. Unter den rekurrenten chromosomalen Alterationen trat die Translokation t(14;18)(q32;q21) am häufigsten auf und war insbesondere bei follikulären Lymphomen Grad 1 und 2, in etwas geringerem Maße auch bei FL Grad 3A anzutreffen. Diese Translokation scheint also in einem frühen Stadium der B-Zell-Entwicklung aufzutreten und führt primär zu einem höher differenzierten (zentrozytenreichen) Lymphom. Die t(14;18) bedingt zumeist eine Überexpression des BCL-2 Gens, die sich auch immunhistochemisch nachweisen lässt und diagnostische Verwendung findet. Das BCL-2 Protein ist daher von Nutzen für die Unterscheidung neoplastischer von reaktiven Follikeln, nicht aber, um follikuläre von anderen „low grade“ B-Zell Lymphomen zu unterscheiden. Die sekundären Alterationen charakterisieren bestimmte undifferenzierte Stadien mit hohem Blastenanteil und einer hohen mitotischen und proliferativen Aktivität. In zytogenetischen Untersuchungen von überwiegend diffus wachsenden FL konnte keine Translokation t(14;18)(q32;q21) nachgewiesen werden. Die identische Morphologie dieser Lymphome und die identischen Veränderungen auch auf genetischer Ebene deuten auf die nahe Verwandtschaft der überwiegend diffus wachsenden FL mit den typischen Keimzentrums-lymphomen hin. Es handelt sich jedoch um eine eigenständige Identität, die differenzierten Keimzentrumslymphome, die wahrscheinlich aufgrund des Fehlens einer t(14;18)(q32;q21) ein primär und ausgeprägt diffuses Wachstumsmuster aufweisen.
Anti-EGFR targeted therapy is a potent strategy in the treatment of metastatic colorectal cancer (mCRC) but activating mutations in the KRAS gene are associated with poor response to this treatment. Therefore, KRAS mutation analysis is employed in the selection of patients for EGFR-targeted therapy and various studies have shown a high concordance between the mutation status in primary CRC and corresponding metastases. However, although development of therapy related resistance occurs also in the context of novel drugs such as tyrosine kinase-inhibitors the effect of the anti-EGFR treatment on the KRAS/BRAF mutation status itself in recurrent mCRC has not yet been clarified. Therefore, we analyzed 21mCRCs before/after anti-EGFR therapy and found a pre-/posttherapeutic concordance of the KRAS/BRAF mutation status in 20 of the 21 cases examined. In the one discordant case, further analyses revealed that a tumor mosaicism or multiple primary tumors were present, indicating that anti-EGFR therapy has no influence on KRAS/BRAF mutation status in mCRC. Moreover, as the preselection of patients with a KRASwt genotype for anti-EGFR therapy has become a standard procedure, sample sets such ours might be the basis for future studies addressing the identification of potential anti-EGFR therapy induced genetic alterations apart from KRAS/BRAF mutations.
Retinoic acid pathway activity in Wilms tumors and characterization of biological responses in vitro
(2011)
Background: Wilms tumor (WT) is one of the most common malignancies in childhood. With current therapy protocols up to 90% of patients can be cured, but there is still a need to improve therapy for patients with aggressive WT and to reduce treatment intensity where possible. Prior data suggested a deregulation of the retinoic acid (RA) signaling pathway in high-risk WT, but its mode of action remained unclear. Results: The association of retinoid signaling and clinical parameters could be validated in a large independent tumor set, but its relevance in primary nephrectomy tumors from very young children may be different. Reduced RA pathway activity and MYCN overexpression were found in high risk tumors as opposed to tumors with low/ intermediate risk, suggesting a beneficial impact of RA especially on advanced WT. To search for possible modes of action of retinoids as novel therapeutic options, primary tumor cell cultures were treated in vitro with all-trans-RA (ATRA), 9cis-RA, fenretinide and combinations of retinoids and a histone deacetylase (HDAC) inhibitor. Genes deregulated in high risk tumors showed opposite changes upon treatment suggesting a positive effect of retinoids. 6/7 primary cultures tested reduced proliferation, irrespective of prior RA signaling levels. The only variant culture was derived from mesoblastic nephroma, a distinct childhood kidney neoplasm. Retinoid/HDAC inhibitor combinations provided no synergistic effect. ATRA and 9cis-RA induced morphological changes suggestive of differentiation, while fenretinide induced apoptosis in several cultures tested. Microarray analysis of ATRA treated WT cells revealed differential expression of many genes involved in extracellular matrix formation and osteogenic, neuronal or muscle differentiation. The effects documented appear to be reversible upon drug withdrawal, however. Conclusions: Altered retinoic acid signaling has been validated especially in high risk Wilms tumors. In vitro testing of primary tumor cultures provided clear evidence of a potential utility of retinoids in Wilms tumor treatment based on the analysis of gene expression, proliferation, differentiation and apoptosis.
Background
Investigation of the expression of an intestinal stem cell marker in esophageal adenocarcinomas (EAC) with and without Barrett's Esophagus (BE), with respect to a cancer stem cell (CSC) hypothesis.
Materials and methods
Expression of a putative intestinal stem cell marker LgR5 was analyzed in esophageal cancer specimen (n = 70: 41 EAC with BE, 19 EAC without BE, and n = 10 esophageal squamous-cell carcinomas, ESCC) and in the adenocarcinoma cell line OE-33. Ki-67 and Cdx-2 were co-labelled with LgR5 in double staining experiments. Immunhistochemical expression results were confirmed by RT-PCR and correlated with tumor stage and five-year survival rates.
Results
LgR5was found expressed in 35 of 41 (85%) EAC with BE and in 16 of 19 (81%) EAC without BE. By contrast, LgR5 was not found to be expressed in ESCC. Quantification of immunolabeling showed 15% LgR5+ cells in EAC with BE, 32% LgR5+ cells in adjacent BE and 13% in EAC without BE. Immunofluorescence double staining experiments with LgR5 and Ki-67 revealed a subpopulation (~5%) of proliferating LgR+/Ki-67+ cells. On mRNA-level, expression of LgR5 was higher in BE in comparison to EAC (p = 0.0159). High levels of LgR5 expression in BE associated EAC were associated with poorer survival in univariate analysis.
Conclusion
The stem cell marker LgR5 is expressed in EAC, irrespective of association with BE, and appears to have negative impact on survival. The subset of proliferating LgR5+ cells (<5%) might resemble rapidly cycling CSCs, which needs to be substantiated in further investigations.