Oncotest GmbH Institute for Experimental Oncology

Freiburg, Germany

Oncotest GmbH Institute for Experimental Oncology

Freiburg, Germany
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Singh M.K.,Albert Ludwigs University of Freiburg | Singh M.K.,University of Tübingen | Ren F.,Albert Ludwigs University of Freiburg | Giesemann T.,Albert Ludwigs University of Freiburg | And 9 more authors.
Plant Journal | Year: 2013

Bacterial protein toxins which modify Rho GTPase are useful for the analysis of Rho signalling in animal cells, but these toxins cannot be taken up by plant cells. We demonstrate in vitro deamidation of Arabidopsis Rop4 by Escherichia coli Cytotoxic Necrotizing Factor 1 (CNF1) and glucosylation by Clostridium difficile toxin B. Expression of the catalytic domain of CNF1 caused modification and activation of co-expressed Arabidopsis Rop4 GTPase in tobacco leaves, resulting in hypersensitive-like cell death. By contrast, the catalytic domain of toxin B modified and inactivated co-expressed constitutively active Rop4, blocking the hypersensitive response caused by over-expression of active Rops. In transgenic Arabidopsis, both CNF1 and toxin B inhibited Rop-dependent polar morphogenesis of leaf epidermal cells. Toxin B expression also inhibited Rop-dependent morphogenesis of root hairs and trichome branching, and resulted in root meristem enlargement and dwarf growth. Our results show that CNF1 and toxin B transgenes are effective tools in Rop GTPase signalling studies. © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd.

Schneider M.,University of Heidelberg | Schneider M.,Oncotest GmbH Institute for Experimental Oncology | Huber J.,University of Heidelberg | Hadaschik B.,University of Heidelberg | And 3 more authors.
BMC Cancer | Year: 2012

Background: Isolation and characterization of tumourigenic colon cancer initiating cells may help to develop novel diagnostic and therapeutic procedures.Methods: We characterized a panel of fourteen human colon carcinoma cell lines and their corresponding xenografts for the surface expression of potential stem cell markers CD133, CD24, CD44, CDCP1 and CXCR4. In five cell lines and nine xenografts, mRNA expression of these markers was determined. Tumour growth behaviour of CD133+, CD133- and unsorted SW620 cells was evaluated in vivo.Results: All five putative stem cell markers showed distinct expression patterns in the tumours examined. Two patient-derived cell lines highly expressed CD133 (> 85% of positive cells) and three other cell lines had an expression level of about 50% whereas in long-term culture based models CD133 expression ranged only from 0 to 20%. In 8/14 cell lines, more than 80% of the cells were positive for CD24 and 11/14 were over 70% positive for CD44. 10/14 cell lines expressed CDCP1 on ≥ 83% of cells. CXCR4 expression was determined solely on 94 L and SW480.Analyses of the corresponding xenografts revealed a significant reduction of cell numbers expressing the investigated surface markers and showed single cell fractions expressing up to three markers simultaneously.Statistical analysis revealed that the CXCR4 mRNA level correlates negatively with the protein expression of CD133, CD44, CD24 and CDCP1 in cell lines and xenografts.A lower differentiation grade of donor material correlated with a higher CDCP1 mRNA expression level in the respective tumour model.In vivo growth behaviour studies of SW620 revealed significantly higher take rates and shorter doubling times in the tumour growth of CD133 positive subclones in comparison to the unsorted cell line or CD133 negative subclones.Conclusions: Our data revealed correlations in the expression of surface markers CD44 and CD24 as well as CD44 and CDCP1 and strongly suggest that CD133 is a stem cell marker within our colon carcinoma panel. Further studies will elucidate its role as a potential therapeutic target. © 2012 Schneider et al; licensee BioMed Central Ltd.

Adla S.K.,TU Braunschweig | Sasse F.,Helmholtz Center for Infection Research | Kelter G.,Oncotest Institute for Experimental Oncology GmbH | Fiebig H.-H.,Oncotest Institute for Experimental Oncology GmbH | Lindel T.,TU Braunschweig
Organic and Biomolecular Chemistry | Year: 2013

The marine natural product flustramine A was synthesised via oxidative cyclisation of Nb-methylated 1-prenyl-2-tert-prenyl-6-bromotryptamine and subsequent reduction of the resulting amidinium salt. Only the tert-prenyl group migrated, whereas the 1-prenyl group remained in place. Interestingly, the 2-tert-prenylated precursor revealed to be the biologically most active of our entire series of 21 compounds. Required for cytotoxicity and antimicrobial activity was the presence of a non-cyclised tryptamine side chain carrying a free secondary amine, whereas the presence of a 6-bromo substituent did not enhance cytotoxicity. In a panel of 42 human tumor cell lines, most sensitive were the lung and mammary cancer cell lines LXFA629L (IC50 1.9 μM) and MAXF401NL (IC50 2.4 μM), respectively. In a serial dilution assay, satisfying IC50 values of 5.9 μM against Micrococcus luteus and 7.7 μM each against Mycobacterium phlei were determined for N b-methyl-1-prenyl-2-tert-prenyl-6-bromotryptamine. This journal is © The Royal Society of Chemistry.

Telle W.,TU Braunschweig | Kelter G.,Oncotest Institute for Experimental Oncology GmbH | Fiebig H.-H.,Oncotest Institute for Experimental Oncology GmbH | Jones P.G.,TU Braunschweig | Lindel T.,TU Braunschweig
Beilstein Journal of Organic Chemistry | Year: 2014

The marine natural product malevamide D from the cyanobacterium Symploca hydnoides was synthesized for the first time. The final peptide coupling linked the dolaisoleuine and dolaproine subunits. The phenyl group of malevamide D was also functionalized with a photoreactive diazirine moiety, which was carried through seven reaction steps. Comprehensive assessment of the cytotoxicity in a panel of 42 human cancer cell lines revealed a geomean IC70 value of 1.5 nM (IC50 0.7 nM) for malevamide D, whereas the photoreactive derivative proved to be less active by a factor of at least 200. COMPARE analysis indicated tubulin interaction as likely mode of action of malevamide D. © 2014 Telle et al; licensee Beilstein-Institut.

Hentschel F.,TU Braunschweig | Raimer B.,TU Braunschweig | Kelter G.,Oncotest Institute for Experimental Oncology GmbH | Fiebig H.-H.,Oncotest Institute for Experimental Oncology GmbH | And 2 more authors.
European Journal of Organic Chemistry | Year: 2014

The first photoreactive derivative of the cytotoxic marine natural product psammaplin A (1) has been synthesized by appending 1-azi-2,2,2-trifluoroethyl moieties at both benzene rings. Photopsammaplin 8 exhibited anticancer activity in vitro with a geomean IC50 value of 1.4 μM in a panel of 42 human cancer cell lines. Particularly sensitive cell lines were found among bladder, lung, mammary, and prostate cancer as well as melanoma (MEXF 276), with IC50 values ≤ 0.6 μM. In a COMPARE analysis, diazirine-functionalized photopsammaplin exhibited a very similar cytotoxicity profile to that of the natural product 1. Furthermore, photopsammaplin 8 was found to be a potent HDAC inhibitor (IC50 35 nM) and can be considered as a suitable candidate for photoaffinity labeling, which may assist in the identification of new targets of psammaplin A. Irradiation experiments with a model diazirine-functionalized α-hydroxy iminoester in dichloromethane in the presence or absence of acetic acid and methanol revealed the stability of the oxime unit and confirmed that reactions took place solely at the carbene center. The isolation of triplet and singlet photoproducts is consistent with DFT calculations [B3LYP 6-311G (2d,2p)] showing that, in polar solvents, the triplet-singlet gap is very small. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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