Selective Modulation of Hedgehog/GLI Target Gene Expression by Epidermal Growth Factor Signaling in Human Skin Maria Kasper1, Michaela Hanneder1, Gerhard Regl1, Stefan Klingler1, Graham W. Neill3, Harald Schnidar1, Leander Blaas2, Carmen Schmid1, Cornelia Hauser-Kronberger2, Michael P. Philpott3 and Fritz Aberger1 1 Department of Molecular Biology, University of Salzburg, Austria 2 Department of Pathology, St. Johann´s Hospital, Paracelsus Medical School Salzburg, Austria 3 Center for Cutaneous Research, Barts and The London Queen Mary’s School of Medicine & Dentistry, University of London, UK Hedgehog/GLI signaling plays a critical role in epidermal development and Basal Cell Carcinoma. Here we provide evidence that Epidermal Growth Factor (EGF) signaling modulates the target gene specificities of GLI transcription factors in epidermal cells. We identified by DNA-array analysis a set of 21 genes, whose transcriptional regulation by GLI1 is dependent on parallel EGF signaling. Promoter analysis of a subset of GLI/EGF regulated genes suggests convergence of EGF and HH/GLI signaling at the level of promoters of selected direct GLI target genes. Synergistic activation of GLI/EGF target genes required ERK1/2 activity, showing that EGF signals via RAF/MEK/ERK to cooperate with GLI proteins in target gene regulation. Co-expression of GLI/EGF targets, EGFR and activated ERK1/2 in human anagen hair follicles argues for a cooperative role of EGF and HH/GLI in specifying the fate of Outer Root Sheath (ORS) cells. We also show that EGF neutralizes GLI-mediated induction of epidermal stem cell marker expression. The results suggest that EGF modulates GLI target gene specificity in ORS cells, which is likely to play an important regulatory role in epidermal stem cell activation, ORS specification and hair growth.

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Characterization of a putative tumor stem cell in malignant brain tumors D. Sommerlad2, A. Schaenzer2, A. Abera2, S. Seidel2, M. Nister3, K.H. Plate2, J. Frisén1, T. Acker1,2 1Karolinska Institute, CMB, Stockholm, Sweden; 2Edinger Institute, Neuropathology, Frankfurt, Germany; 3Karolinska Hospital, CCK, Stockholm, Sweden Self-renewal and multi-potency are by definition essential characteristics of stem cells. Organ-specific stem cells have been identified in a number of tissues including the hematopoietic system, intestine, skin and the central nervous system. On the other hand, dysregulated self-renewal is a feature of tumor growth. The aberrant activation or dysregulation of organotypic stem cell pathways has been classically associated with tumor growth and progression. Indeed several recent publications point to the existence of a tumor stem cell and suggest remarkable parallels between stem cell and tumor cell biology. The capacity of cell to exclude fluorescent dyes such as Hoechst 33342 correlates with the "stemness" of the cell, thus, characterizing the side population (SP). SP can be isolated by FACS analysis from different tissues and is highly enriched for cells with stem cell function. Using this method a SP encompassing 0,1%-1,1% of all cells could be identified in different glioblastomas, the most malignant brain tumor. The SP fulfilled tumor stem cell characteristics, i.e. SP cells were able to self-renew, gave rise to a more differentiated progeny and formed tumors in vivo. Both, growth factors (e.g. EGF, FGF) as well as transcription factors (HIF -hypoxia inducible factor) modulated the size of the SP. In analogy to neural stem cells, glioblastoma cells when cultured in serum-free neurosphere medium grew as tumor spheres over several passages. Upon induction of differentiation they expressed glial and neuronal markers. Thus, several lines of evidence suggest that glioblastomas harbor a subpopulation of tumor cells with stem cell characteristics giving rise to a heterogeneous progeny. Further understanding to what degree tumor stem cells and organ-specific stem cells make use of related signalling mechanisms to regulate self-renewal, proliferation and differentiation may provide deeper insights into tumor pathophysiology and may, in addition, help as a "Spin-off" to understand processes of physiological stem cell homeostasis.

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MafB deficiency results in myeloid biased repopulation advantage of hematopoietic stem cells Taro FUKAO; Sandrine SARRAZIN; Athar AZIZ; Louise M KELLY; Estelle DUPREZ; Noushine MOSSADEGH; Michael Herbert SIEWEKE, PhD Parc scientifique de Luminy, Centre d'Immunologie de Marseille-Luminy CNRS-INSERM-Université de la Méditerranée Campus de Luminy, Case 906, 13288 Marseille Cedex 09, France Hematopoietic stem cells (HSC) continuously regenerate blood cells of all lineages but little is known about the mechanisms that assure the balanced production of lineage specific progeny. Here we report that the monocyte transcription factor MafB is expressed in HSC and selectively restricts the potential for myeloid cell generation. Despite normal numbers of HSC and progenitors in MafB-/- fetal livers, competitive transplantations revealed normal contribution of MafB-/- HSC to T-cell controls but a strong capacity to replace wild type cells in the myelo-monocytic lineage, without transgressing lineage boundaries. This lineage biased repopulation advantage was progressive, maintained long term and enhanced through serial transplantation. Furthermore, MafB deficiency caused strongly accelerated cell division of c-kit+, sca-1+, lin- HSC in M-CSF culture but not under T-cell differentiation conditions. Together this suggests that MafB restricts the intrinsic potential of HSC to generate myeloid progeny without compromising self-renewal and independently of the final number of mature cells.

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Frizzled-9 and CDCP1 (CD318) Expressing Placenta and Bone Marrow MSC Differentiate into Multiple Germ Line Lineages Venkata Lokesh Battula1, Andreas M. Boehmler1, Sabrina Treml1, Petra Bareiß2, Ingrid Albert3, Sigrid Hojak3, Lothar Just2, Thomas Skutella2, Hans Kiefer3, Hans-Jörg Bühring1

1) University Clinic of Tübingen, Department of Internal Medicine II, Division of Hematology, Immunology, Oncology and Rheumatology, Tübingen, Germany 2) University of Tübingen, Institute of Anatomy, Department of Experimental Embryology,

Division of Tissue Engineering, Tübingen, Germany 3) m-phasys GmbH, Tübingen, Germany Mesenchymal stem cells (MSC) can be differentiated into several cell types including bone, cartilage, muscle, stromal cells, tendon and connective tissue. Conventionally, MSC are generated by plating primary cells into culture flasks and selecting plastic-adherent cells with fibroblast-like morphology. Using a novel approach, MSC were generated by culture of human BM and placenta on gelatine-coated plates in the presence of bFGF. These cells expressed conventional mesenchymal markers like CD13, CD105, and CD164 in addition to CUB-domain-containing protein 1 (CDCP1; CD318). In contrast to conventionally prepared MSC, they also expressed the embryonic stem cell markers SSEA-4 and oct-4, theprogenitor marker nestin and the wnt receptor Frizzled-9 (FZD9). Further differentiation of these cells on ultra-low adherent culture dishes resulted in the appearance of large cell aggregates with embryoid body-like structures. Culture of these cells in insulin, transferrin and selenite (ITS) containing medium resulted in the appearance of adherent cells with fibroblast-like morphology. When cultured under appropriate conditions, these cells gave rise to functional adipocytes and osteoblast-like cells (mesoderm), glucagon expressing pancreatic islet-like cells (endoderm), as well as class III β-tubulin expressing neuron-like and GFAP expressing astrocyte-like cells (ectoderm). In summary, using a new protocol we show that MSC derived from neonatal placenta and adult bone marrow express the novel markers Frizzled-9 and SSEA-4 and are able to differentiate into multiple germ line lineages.

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Mesenchymal stem cells (MSCs) and angiogenesis in pancreatic carcinoma Beckermann, B1., Kallifatidis, G1., Wenger, T.1, Schubert, M.2, Krause, U.2, Su, Y.3, Friess, H.3, Herr, I.1 and Büchler, P.31Molecular Urooncology, DKFZ, Heidelberg, Germany, 2Policlinic and 3Department of Surgery, University of Heidelberg, Germany Aims: Homing of manipulated mesenchymal stem cells (MSCs) to glioma cells or of embryonal progenitor cells to lung metastases in mouse xenograft models has been demonstrated in recent reports. Also, endothelial progenitor cells are known to participate in formation of new tumour blood vessels induced by hypoxic conditions in expanding tumours. MSCs may contribute to formation of tumour stroma and tumour blood vessels and thus be ideal vehicles for the targeted transfer of therapeutic genes. Methods: MSCs are selected from intraoperatively harvested bone marrow and tumour-specific migration in vitro to tumour cell lines and in vivo to tumour xenografts on nude mice is detected. For in vivo detection, MSCs are marked by lentiviral GFP. Transdifferentiation to tumour stroma and endothelial-like cells upon homing in tumours is analyzed by surface staining. Involvement of MSCs in formation of blood vessels is examined in vitro by spheroid assays and in vivo by staining of cryosections. For therapeutic intervention, lentiviral vectors are constructed to inhibit angiogenesis by downregulation of VEGF- or HIF1-alpha using the RNAi technology. Results: Tumour-specific in vitro homing of non-manipulated MSCs selected from bone marrow of patients was found. Lentiviral GFP-control vector transduced to MSCs resulted in a transfection efficiency of 20% which could be enhanced to 90% by flow cytometry sorting. Injection of lentiviral GFP-expressing MSCs into the tail vein of nude mice xenografted with an orthotopic growing pancreatic tumours demonstrated in vivo homing to the tumour cells. Lentiviral vectors efficiently prevented hypoxia-induced VEGF or HIF1-alpha expression when transduced to tumour cell lines. Our actual work focuses on transdifferentiation of MSCs, involvement of MSCs in tumour angiogenesis and prevention of tumour growth by transduction of MSCs with lentiviral VEGF-or HIF1-alpha RNAi constructs. Conclusions: Autologous MSCs may be a powerful tool for transfer of therapeutic genes into tumours, e.g. for specific inhibition of tumour angiogenesis.

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Signaling mechanisms in human embryonic stem cells (hESC) Daniel Besser Max-Delbrück-Center, Robert-Rössle-Straße 10, 13125 Berlin Human embryonic stem cells will remain undifferentiated or undergo differentiation when grown in conditioned or non-conditioned medium, respectively. The factors and signaling events that control the maintenance of the undifferentiated state are not well characterized. Our data show that nodal and the inhibitors of Nodal signaling, lefty-A and lefty-B, are down-regulated very early upon differentiation. High expression of these genes in undifferentiated cells is maintained by activation of the transcription factor Smad2/3, downstream of the activin-linked kinases (ALK) 4/5/7. Activation of ALK4/5/7 with Activin A and inhibition of these receptors by the kinase inhibitor SB-431542 leads to regulation of the Smad2/3 activity and of the expression of nodal, lefty-A and lefty-B. In addition, when cells are maintained undifferentiated by treatment with the GSK3- inhibitor, BIO, high expression of these genes also requires activation of ALK4/5/7. Conversely, BMP signaling leading to Smad1/5/8 activation is blocked in undifferentiated cells and becomes activated upon differentiation. Moreover, we have shown that the activation of the Activin/Nodal pathway is required for the maintenance of the undifferentiated state in human embryonic stem cells, but its activation alone is not sufficient to maintain the cells undifferentiated.

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An Optical Method for Nanostructure Analysis of Specific Gene Domains for use in Cancer and Stem Cell Research Udo Birk, Jutta Schwarz-Finsterle, David Baddeley, Georg Hildenbrand, Alexander Rapp, Michael Hausmann, Christoph Cremer. Kirchhoff Institute for Physics, University of Heidelberg, D-69120 Heidelberg In order to extract information on gene activity “in vivo”, it would be highly advantageous to derive the degree of transcriptional activation directly from a (light-)microscopical visualization of structural changes in the gene domain. Spatially Modulated Illumination (SMI) Microscopy provides a powerful tool to measure the extension of specific gene domains after fluorescence labeling, thus allowing to detect differences in the conformational changes of these genes. The method of SMI nanostructure analysis is based on the fact that the modulation of the axial Point Spread Function (PSF) of the SMI microscope is “disturbed” by the size of the object. Using theoretical models, this “disturbance” of the modulation can be used to determine the original (axial) extension of the object. It has been shown that a strong correlation exists between the “disturbance” of the modulation and the object’s axial extension, and true sizes (diameters) could be measured for fluorescent nano-spheres down to about 40nm. Furthermore, measurements of the axial extensions of specific gene regions (e.g. p53 or cmyc) indicated that the volume occupied by a specific gene depends on parameters additionally to the length of its DNA sequence only. By measuring the axial extension of the volumes occupied by the abl gene region in CML blood cells, a first step towards the microscopical detection of blood cancer related alterations correlated to the induction of translocations has been successfully performed. These measurements in principle allow the detection of structural changes of breakpoint regions in an early stage or closely related to the formation of the Philadelphia Chromosome or other translocations. Moreover, in quantitative molecular cytogenetics this “nanosizing” method allows a variety of applications. Examples are: gene expression analysis; gene copy analysis in 3D-intact nuclei; transcription factories analysis; protein cluster analysis. An important practical feature of the SMI-nanosizing method described is that such studies may be conducted using conventional object slides for specimen preparation, which can be seen as an important methodological advantage in contrast to other high-resolution microscopy techniques.

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Migrating Cancer Stem Cells - An integrated concept of malignant tumour progression Thomas Brabletz, Simone Spaderna, Falk Hlubek, Otto Schmalhofer, Thomas Kirchner, Andreas Jung Dept. of Pathology, Univ. of Erlangen, Germany Most colorectal adenocarcinomas (CRC) have mutations in the APC gene leading to overexpression of the Wnt-pathway effector β-catenin. By acting as a transcriptional activator, nuclear β-catenin is decisively involved in two fundamental processes in embryonic development: epithelial to mesenchymal transition (EMT) and generation of stem cells (e.g. in intestine development). We have previously shown, that nuclear β-catenin accumulates in tumor cells at the invasive front undergoing an EMT. The amount of these cells strongly correlates with clinical outcome and metastasis formation. In contrast tumor cells in central tumor areas are differentiated and often show membranous, E-cadherin-bound β-catenin. Strikingly, also metastases show again a differentiated phenotype lacking nuclear β-catenin, indicating a regulatory role of the tumor environment for malignant progression. Based on the developmental functions of β-catenin and our data we propose, that the EMT-associated tumor cells at the invasive front act as "migrating cancer stem cells" which can re-differentiate and, depending on the range of dissemination, give raise to the primary carcinoma or metastases (1). In support of this hypothesis, we defined β-catenin target genes overexpressed in invading tumor cells (by using reporter assays, EMSA, ChIP, siRNA techniques, in situ hybridisation, immunohistochemistry and animal models): 1. A stem cell phenotype is supported by the β-catenin target genes hTERT and survivin. 2. Furthermore we identified target genes like uPA, MT1-MMP, and laminin5-γ2 chain, which synergize to exert EMT and promigratory activity. We suggest that both primary tumors and metastases are derived from a pool of EMT-associated "migrating cancer stem cells" at the tumor host interface, which are defined by strong nuclear β-catenin accumulation and expression of its target genes. This gives these cells a feature, which drives malignant tumor progression including metastasis: the unusual combination of abilities allowing simultanously to migrate and behave as a cancer stem cell.

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Wnt-deficient embryonic stem cells as a tool for understanding the role of Wnts in the dopaminergic development Vítězslav Bryja, Sonia Bonilla, Lukáš Čajánek, Clare L. Parish, Catherine M. Schwartz*, Yongquan Luo*, Mahendra S. Rao* & Ernest Arenas; Laboratory of Molecular Neurobiology, MBB, Karolinska Institutet, Stockholm, Sweden, S-171 77 & *Gerontology Research Center, Stem Cell Biology Unit, Laboratory of Neurosciences, NIA, NIH, Baltimore, MD, 21224-6825 Embryonic stem cells (ESC) represent unique source of cells for the cell replacement therapy in several diseases including Parkinson’s disease. The ability of ESCs to differentiate into many cell types makes them an ideal tool for the analysis of development in vitro. The process of ESC derivation is often very inefficient and requires high specialization, training and expertise. In order to circumvent these limitations we developed a simple and efficient protocol based on the use of commercially available products. Here we used that protocol to derive ESCs deficient in Wnt-1, Wnt-5a, and Lrp6 with a 50-75% efficiency. Using these new transgenic ESCs we study the function of Wnt signalling pathway in the development of dopaminergic neurons and present the characterization of these cell lines. In summary, we provide a simplified and improved protocol to derive mESCs that can be easily applied to other strains deficient in components of the Wnt pathway. By promoting dopaminergic differentiation in ESCs we aim at understanding the role of Wnts in the development of dopaminergic neurons. These findings will help us to develop new strategies aimed at improving cell replacement therapy for Parkinson’s disease using Wnts and their inhibitors.

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Focused Laser Beams for Stem Cell Purification and Clonal Expansion Buchstaller, S. Soria-Lopez, S. Sagebiel, R. Huss. Institute of Pathology, University of Munich, Munich, Germany. Stem cells have the potential to differentiate into specialized cells from various lineages. Differentiation is often associated with changes in morphology and cell surface marker expression. To understand the intrinsic molecular events associated with differentiation, it is important to gain pure populations of cells and to analyze their behavior and characteristics. The selection and isolation of cells with specific features also stand at the center of stem cell therapy and tissue engineering. Heterogeneity of cell preparations poses a serious problem to the molecular analysis and therapeutic use of stem cells. We used “Laser Microdissection and Pressure Catapulting” (LMPC, P.A.L.M. Microlaser Technologies) for the isolation of life stem cells according to cell morphology and marker expression. We first isolated single cells from 1) murine embryonic carcinoma cell lines, 2) murine adult stem cell lines and 3) preparations of human bone marrow mononuclear cells. The recultivated clonal cells expand rapidly, express the same stem cell-specific markers as the originating cells and maintain the same morphology. We then proceeded to sort out and clonally expand single adherent fibroblast-like CD29+/CD44+ mesenchymal stem cells from a complex mixture of bone marrow-derived cells. In a different set of experiments we separated undifferentiated embryonic carcinoma cells from their differentiated progeny after treatment with all-trans retinoic acid. Cells exhibiting a neuronal morphology including neurites and growth cones were isolated with LMPC, recultivated, and analyzed for the expression of the growth-associated molecule GAP-43 and activity of L-type calcium channels. Applied in a high throughput fashion, coupled to single cell manipulation and high content single cell analysis, LMPC will prove itself as an important tool in basic research, drug development, and regenerative medicine.

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BMP-2 Dependent Integration of Mouse Adult Subventricular Stem Cells Transplanted into the Neural Tube of Chick and Quail Embryos C. Busch, M. Oppitz, M. Sailer, U. Drews Objective. BMP-2 induces neural crest cell fates in adult subventricular stem cells in vitro. The objective of the experiments was to test the effect of BMP-2 in the microenvironment of the embryonic neural tube. Methods: Adult neural stem cells were isolated from the subventricular zone of three weeks old male GFP mice and grown in vitro as neurospheres with bFGF. 24h prior to transplantation into the neural tube of 2 day chick or quail embryos half of the cultures were treated with BMP-2. In addition to life fluorescence, GFP, HNK1 and QCPN immunohistochemistry and in situ hybridization with mouse specific mL1 were performed in paraffin and cryostat sections. Results: Non-BMP-2 treated neurospheres did not integrate into the neuroepithelium. BMP-2 treated neurospheres formed close contacts with the dorsal neuroepithelium corresponding to the neural crest area. Murine cells emigrated and were found in the in the roof of the neural tube, in the dorsal neuroepithelium and among emigrating neural crest cells adjacent to the neural tube. Conclusion: The results show that BMP-2 pre-treatment enables neurospheres to emigrate into the embryonic neuroepithelium. Specific neural crest cell migration patterns were not observed.

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Effect of GDF-15 in neural precursors development. Carrillo-García, C.; Strelau, J.; Unsicker, K. and Ciccolini, F. Neural stem cells (NSCs) are defined by their ability to self-renew and to give rise to the three main cell-types of the central nervous system. During development NSCs modify their properties, such as differentiation potential and ability to respond to growth factors. At early stages of development, NSCs tend to generate greater amount of neurons, whereas late development NSCs are biased toward glia generation. Late stem cells are responsive to EGF, whereas early progenitor cells are mitotically responsive to FGF-2 but not to EGF. Extrinsic signals have been shown to modulate EGFR expression and the differences in the level of EGFR expression determine how progenitor cells respond to EGF at specific stages of development. (Ciccolini et al 1998; Lillien et al 2000; Ciccolini, 2001; Ciccolini et al, 2005) GDF-15, a member of the TGF-β superfamily, is involved in cellular processes such as inflammation, apoptosis/survival, and progression of a number of cancers. It has been shown that it reduces cell adhesion and consequently promotes cell detachment and tumor dissemination. (Liu et al, 2003; Shim et al; 2005) GDF-15 is highly expressed in choroid plexus epithelium of all ventricles in rat and is secreted into the cerebrospinal fluid (Schober et al. 2001). Here we show that GDF-15 is highly expressed in neurogenic areas of the mouse brain and that its expression is higher in isolated progenitor cells. Furthermore we have tested the possibility that GDF-15 plays a role on precursor proliferation and differentiation.

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Changes of antigen expression and EGFR levels in SVZ precursors by modulation of neurogenesis. Francesca Ciccolini1, Claudia Mandl1, Gabriele Hölzl-Wenig1, Kertin Horsch2, and Volker Eckstein2. 1Department of Neurobiology, Interdisciplinary Center for Neurosciences (IZN), University of Heidelberg, Im Neuenheimer Feld 364, 69120 Heidelberg, Germany. 2Medizinische Klinik V, University of Heidelberg, Im Neuenheimer Feld 410, 69120 Heidelberg, Germany. The subventricular zone (SVZ) is the main site of precursor proliferation and neurogenesis in the adult brain. It has been suggested that the coordinated proliferation and differentiation of three precursor types drives the process of neurogenesis in this area. However it is not clear if these precursor populations represent different cell types or different cell status and how they relate to the main SVZ stem cell. Here we use lineage specific markers and transgenic mice that express GFP under the control of a neuronal specific promoter to isolate different subsets of SVZ progenitors by flow cytometry and analyse their ability to proliferate and differentiate by clonal assay. Using this approach we found that clone forming activity in the SVZ of adult mice is mostly associated with PSA-NCAM+ cells that express high levels of EGFR (PSA-NCAM+/EGFRhigh) and allow intermediate levels of transcription from a neuronal-specific promoter. Within this cell population less than 5% cells expressed Lex-ssea1 and only 15% mCD24 antigen. The remaining stem cell activity was associated with PSA-NCAM cells expressing low EGFR levels (PSA-NCAM+/EGFRlow) and that were Lex- ssea1 and mCD24 immunonegative. We next analysed the expression of these antigens in conditions of neurogenesis modulation. We found that neurogenesis correlates with down-regulation of Lex- ssea1 and mCD24 expression and an increase in the pool of PSA-NCAM+/EGFRlow precursors cells. These results indicate that in SVZ precursors expression of these antigens is dynamically regulated and does not define distinct precursor types.

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First measurement of integrin VLA-4 activation by CXCL12/SDF-1 on a living cell and at a single-molecule level: Nanobiotech and the new concept of “metastasizing stem cells” in cancer Robert H. Eibl Institute of Pathology, Technical University of Munich, Germany Atomic force microscopy (AFM) based force spectroscopy can be used for direct measurement and quantification of cell adhesion forces on living cells down to single-molecule interactions. This technique is used here to study the physiologic activation of alpha(4) integrins, e.g. VLA4 by the chemokine CXCL12 (=SDF-1) on living cells expressing the chemokine receptor CXCR4 – recently related to organ-specific metastasis of many cancers. At low physiologic concentration of CXCL12 the rupture force of a single pair of adhesion receptors increases from 40pN to 60-80pN, whereas at higher concentrations of CXCL12 it lowers the rupture forces back to 40pN. This study confirms that arrest chemokines such as CXCL12 can rapidly activate and modulate the affinity of an integrin receptor. To date, this is the first direct measurement of chemokine induced affinity modulation of a single (adhesion) receptor – of any integrin on any cell of any species - as well as of the desensitization of a chemokine receptor, both measured on a molecular level on a living cell. In conclusion, AFM based force spectroscopy has evolved into a completely new pharmacological test for single-molecule interactions on living cells. This technique will be used to elucidate the mechanisms involved in normal and pathological leukocyte homing (adult stem cell homing, Asthma, Multipe Sclerosis, Type I Diabetes), as well as for organ-specific tumor (stem) cell metastasis and the development/optimization of new inhibitors of metastatic cancers.

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Identifying Genes Responsible for Apoptosis-Resistance in Hematopoietic Malignancies Expressing wt p53 by Means of Retroviral Insertional Mutagenesis Afra Engelmann, Wolfgang Deppert and Carol Stocking HPI - Heinrich-Pette-Institut für Experimentelle Virologie und Immunologie an der Universität Hamburg High grade Non-Hodgkin’s Lymphomas (NHL) respond well to classical chemo- or radiation therapies, a fact that correlates well with the observation that wild-type p53 expression is detected in 80 to 95% of tumors, in striking contrast to carcinomas. However, despite the initial good response, long-term survival rates are generally poor in NHL patients, due to the outgrowth of therapy resistant clones and subsequent relapse of the disease. Interestingly, the wild-type p53 status is often maintained in the secondary disease. Defining the mechanisms by which these tumors escape apoptosis while maintaining an intact p53 gene is an important endeavor towards the development of effective therapies. Our goal is to define critical mutations in radiation-resistant clones by analysis of integration sites unique to these tumors. For this work, a mouse model was chosen, in which a lymphoid tumor is induced by Moloney murine leukemia virus (MoMLV) infection. The mouse strain carries an activated human cMYC oncogene under control of the Igλ enhancer, which provides the “first hit” in tumorigenesis. The developing tumors show a phenotype similar to Burkitt’s lymphoma and the majority (∼80%) express wt p53, consistent with the findings in Burkitt’s lymphoma. Significantly, γ irradiation triggers apoptosis in these cells, but cells that escape apoptosis can be detected in transplanted animals at an incidence of <10-4. Viral integration sites in ten independent tumor sets were isolated, sequenced and their chromosomal location mapped using available data bases. Moreover, 35 independent tumors were screened for already known common integration sites. Significantly, integrations upstream of the bmi-1 locus were observed in circa 35% of the independent tumors. Integrations in this locus were observed in both, radiation-sensitive and radiation-resistant tumors, supporting the hypothesis that inhibition of the ARF pathway (in this case by Bmi-1) abrogates p53-mediated apoptosis in response to the myc-expression, but not to genotoxic stimulation. A number of other loci were identified that were shared by both radiation resistant and sensitive tumors and previously shown to cooperate with myc-induced tumorigenesis. However, we also identified two loci, at which integration correlated with the acquisition of radiation resistance. One locus was found in two independently derived tumor-sets and mapped to the 1st intron/promoter region of the gene encoding Bcl-X, an important antiapoptotic member of the Bcl-2 family. Quantitative RealTime RT-PCR has confirmed that this gene is overexpressed in these tumors, thus providing a satisfying “proof-of-principle” to our approach. The second integration mapped downstream of the gene encoding GADD45β, and high levels of expression were observed in γ-radiation-resistant tumors. These results support the recent hypothesis that GADD45β can either induce or block apoptosis, depending on the cellular environment. Demonstrating the importance of this pathway in blocking apoptosis in B-cell malignancies will provide new targets for therapeutic development. These results show that our approach is a powerful tool to identify genes that are responsible for apoptosis resistance in B-cell malignancies.

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Skin epidermis lacking the c-myc gene is resistant to Ras driven tumorigenesis but can re-acquire sensitivity upon additional loss of the p21CIP1 gene Marieke Essers1, Thordur Oskarsson1, Nicole Dubois1, Christelle Dubey1, Catherine Roger1, and Andreas Trumpp1,2

1Genetics and Stem Cell Laboratory, Swiss Institute for Experimental Cancer Research (ISREC) Ch. des Boveresses 155, CH-1066 Epalinges, Switzerland. 2Ecole Polytechnique Fédérale de Lausanne (EPFL), School of Life Sciences, CH-1015 Lausanne, Switzerland The c-myc gene is one of the most frequently mutated oncogenes found in human tumors. C-Myc has been implicated in the regulation of various biological processes including cell cycle progression, cellular growth, differentiation, angiogenesis, immortalization and apoptosis. To assess the normal role of c-myc in epithelial cell types in vivo we utilized a tamoxifen inducible K5::CreERT transgene to delete the c-myc gene in the adult skin epidermis via a conditional Cre/LoxP recombination. Unexpectedly, despite its strong expression in the basal compartment, c-myc is not essential for maintenance of the skin epidermis of the adult mouse. The mutant epidermis appears normal with respect to both proliferation and differentiation. In addition, no selection against c-myc deficient epidermal cells occurred over many months, further confirming that c-myc is dispensable for normal skin homeostasis. Even more surprising, TPA induced hyperproliferation also occurred in a c-myc independent manner. Treatment of the skin with the mutagen DMBA prior to TPA is a classical method to induce papillomas, by selecting for mutations that lead to the dominant activation of the oncogene H-Ras. Interestingly, c-myc deficient epidermis is resistant to this Ras mediated chemically induced tumorigenesis suggesting that progression from epithelial hyperplasia to benign tumor formation requires the endogenous c-myc gene. This is mechanistically linked to p21CIP1 which is induced in tumors by activated Ras-ERK pathway but repressed by c-Myc. Acute elimination of c-myc in established tumors leads to the upregulation of p21CIP1, and females lacking both p21CIP1 and c-Myc re-acquire normal sensitivity to DMBA/TPA induced tumorigenesis. This identifies c-Myc mediated repression of p21CIP1 as a key step for Ras driven epidermal tumorigenesis.

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Glioblastoma multiforme-maintaining cells characterized by heterogeneous cell population regeneration capacity and telomerase-dependent genomic stability Christine Glanz,*† Johan Rebetz,*†‡ Ylva Stewénius,§ Annette Persson,¶ Elisabet Englund,¶ Leif G. Salford,* Bengt Widegren,*†‡ David Gisselsson, § and Xiaolong Fan*†**

From the Rausing Laboratory, Division of Neurosurgery, * Lund University Hospital; Section of Immunology, † Department of Experimental Medical Science, Lund University; Department of Cell and Molecular Biology, ‡ Lund University; Department of Clinical Genetics, § Lund University Hospital; Department of Pathology, ¶ Lund University Hospital; Section of Molecular Medicine and Gene Therapy, Strategic Research Center for Stem Cell Biology and Cell Therapy, Lund University. Glioblastoma multiforme (GBM), the most malignant primary brain tumor in adults, is characterized by morphological, immunophenotypic, and genetic heterogeneity. Recently, isolated CD133+ subpopulations of GBM cells have been identified as brain tumor stem cells (BTSCs) based on tumorigenecity, self-renewal and differentiation capacity. By using freshly isolated GBM cells, we established two serially transplantable xenograft GBM lines in SCID-mice to characterize the regenerative capacity and the genetic background of a BTSC-like population. In all passages, both xenograft GBM lines regenerated phenotypically heterogeneous cell populations including CD133+ cells. Comparison between primary and xenograft GBM cells revealed that only a fraction of the cytogenetically defined primary GBM subclones exhibited tumorigenicity under our conditions, coinciding with reduced morphological heterogeneity in xenograft GBM specimens. Primary and early passage xenograft GBM cells showed a high degree of chromosomal instability and telomere dysfunction. In subsequent passages, clones with dysfunctional telomeres were lost, resulting in cells with a stable karyotype and sustained hTERT expression. Inhibition of telomerase triggered dramatic chromatid separation abnormalities in these cells, resulting in polyploidisation and reduced colony formation capacity. This indicates that GBM maintaining cells are dependent on telomerase activity to preserve genomic integrity and cellular proliferation.

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Human Short-term Repopulating Cells Have Enhanced Telomerase Reverse Transcriptase Expression Marcus Järås1, Anna Edqvist2, Johan Rebetz2, Leif G. Salford3, Bengt Widegren2 and Xiaolong Fan1,2

1Section of Molecular Medicine and Gene Therapy, Lund Strategic Research Center for Stem Cell Biology and Cell Therapy, 2Section of Immunology, 3The Rausing laboratory, Division of Neurosurgery, Lund University Hospital, Lund University, Sweden. Telomerase activity has been suggested to be critically involved in hematopoietic stem cell (HSC) self-renewal. However, it has been unclear whether human HSCs have telomerase activity and how telomerase activity is regulated within the HSC and progenitor pool. Here, we isolated living cord blood (CB) CD34+ cells with upregulated human telomerase reverse transcriptase (hTERT) expression by using a hTERT reporting adenoviral vector encoding destabilized GFP (dGFP) driven by the hTERT promoter and functionally characterized them in comparison to control vector transduced CD34+ cells expressing GFP. Following a two-day serum-free transduction protocol, cells were sorted into a dGFP+ and a GFP+ fraction. Cell cycle analysis revealed that the dGFP+ cells had a greater proportion of cells in S/G2/M phase compared to the GFP+ cells (56±1.8% versus 35±4.3%, p<0.001), and fewer cells in G0 phase (8.1±3.0% versus 20±4.7%, p<0.01). Furthermore, the colony-forming and short-term NOD/SCIDBeta2m-/- mice bone marrow repopulating capacities were similar between the dGFP+ and the GFP+ cells. Interestingly, the dGFP+ cells had a 6-fold lower repopulating capacity in NOD/SCID mice compared to the GFP+ cells and lacked secondary NOD/SCIDBeta2m-/- mice bone marrow repopulating capacity. Thus, upregulation of hTERT expression within the CB HSC pool is accompanied by decreased self-renewal capacity.

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Evaluation of the predictive value of 8 candidate genes on therapy outcome in Laser-Microdissected paraffin-embedded osteosarcoma biopsies Fellenberg J., Zahlten-Hinguranage A., Delling G., Bernd L. Stiftung Orthopädische Universitätsklinik, Heidelberg Background: Osteosarcoma ist the most common primary tumor of the bone, typically affecting the long tubular bones of children and adolescents. The introduction of adjuvant and neoadjuvant chemotherapy markedly improved the outcome with long term relapse free survival rates ranging from 55 to 75%. However, the remainder of patients poorly respond to chemotherapy with an increased risk of relapse and the development of metastasis. Histologic response to chemotherapy is currently the strongest prognostic factor in high-grade osteosarcoma, but it can only be assessed after several weeks of therapy. Thus, detection of chemosensitivity at the time of diagnosis would be of great clinical importance. Aim: Gene expression analysis of eight, recently identified, drug regulated genes in osteosarcoma biopsies and the evaluation of their predictive value by the correlation of gene expression data with clinical parameters. Material and Methods: Paraffin sections from 35 osteosarcoma biopsies were analyzed (18 good responder and 17 poor responder). The response to preoperative chemotherapy was assessed histologically according to the six-grade scale of Salzer-Kuntschik. Tumor cells were isolated by Laser-Microdissection followed by gene expression analysis using quantitative real-time PCR. Results: Out of the 8 genes analyzed, the expression of rhoA, impdh2 and pro1959 was significantly elevated in tumors that poorly respond to chemotherapy (2.1-, 2.3- and 6.0-fold, respectively) (p=0.017, p=0.019 and p=0.012). In addition, increased expression of impdh2 correlated with a poor disease-free survival (p=0.012). A significant negative correlation of gene expression and disease-free as well as overall survival could also be detected for prohibitin, ferritin light-chain and pro2000. Conclusion: This study suggests a possible role of rhoA, impdh2 and pro1859 as prognostic marker for the early detection of chemoresistance in osteosarcomas. In addition, the correlation of prohibitin, ferritin light-chain and pro2000 gene expression with the overall and disease-free survival may indicate a more aggressive malignancy. Together, these candidate markers may be of clinical importance, in order to stratify patients at diagnosis into low and high-risk groups improving the outcome of high-risk patients and minimizing the toxicity of therapy for low-risk patients by means of a risk adapted therapy.

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Regulatory T cell development: Resistance of naïve Treg towards CD95L-mediated apoptosis

B.Fritzsching1*, N.Oberle1*, E.Pauly2*, R.Geffers3, J. Buer3, J.Pöschl2, O.Linderkamp2, P.H. Krammer1 and E.Suri-Payer1 1Division of Immunogenetics, German Cancer Research Center, 2Divison of Neonatology, Children´s Hospital, University of Heidelberg; 3Department for Cell Biology and Immunology, German Research Center for Biotechnology, Braunschweig, Germany.

* these authors contributed equally to this work

Since tumor-induced accumulation of immunosuppressive CD4+CD25+FOXP3+ regulatory T cells (Treg) has been shown to be detrimental in cancer, new insights into mechanisms of Treg elimination are warranted. We have recently shown in vitro that treatment of freshly isolated adult Treg with CD95L has caused apoptosis in a majority of these cells†. However, a minor Treg population remains apoptosis-resistant. This rare Treg population has been isolated. Viable Treg were collected from eight healthy donors (8x 500 ml blood) to yield a sufficient cell amount for RNA-microarray analysis. Gene chip analysis revealed an upregulation of naïve cell markers in the CD95L-resistant Treg cell pool. In contrast, the whole Treg compartment showed a clear upregulation of memory markers. FACS analysis confirmed a selective survival of naïve adult Treg after treatment with CD95L in vitro. We next analyzed genuine naïve Treg cells derived from human cord blood as well as peripheral blood of newborns. 6-color-FACS-analysis including FOXP3 intracellular staining showed that almost all CD4+CD25+FOXP3+ cells in newborns are CD95low. Furthermore, the great majority of these freshly isolated cord blood Treg cells are not killed by CD95L. In contrast, the vast majority of CD4+CD25+FOXP3+cells is CD95high and highly CD95L-sensitive in adult individuals. However, cord blood derived Treg strongly upregulated CD95 after short-time stimulation with anti-CD3/28 mAbs and could be efficiently killed by CD95L in vitro. This functional change of cord blood Treg was paralleled by a rapid upregulation of typical memory cell markers leading to a phenotype similar to the majority of adult Treg. In summary, we show a clear functional difference between naïve and memory Treg that could lead to different survival rates of those two cell populations in vivo. This new observation could be crucial in the planning of interventional therapeutical application of Treg cells. † Fritzsching et.al., Cutting Edge, Journal of Immunology, 2005, 175:32-36.

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Methylation-associated transcriptional silencing of the C/EBPalpha gene in acute myelogenous leukemia Edgar Jost, Nicole do Ó, Stefan Wilop, James G. Herman, Rainhardt Osieka, Oliver GalmMedizinische Klinik IV, Universitaetsklinikum Aachen, RWTH Aachen, Germany The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, MD, USA Differentiation of hematopoietic stem cells and progenitor cells is controlled by a regulatory network that includes various transcription factors. The CCAAT/enhancer binding protein alpha (C/EBPalpha) is a transcription factor implicated in the regulation of myelopoiesis. C/EBPalpha is important for the coordination of cellular differentiation with growth arrest. Specific point mutations of C/EBPalpha have been reported in hematological malignancies, especially in acute myelogenous leukemia (AML). Mutated forms of C/EBPalpha may impair granulocytic differentiation and thus contribute to leukemogenesis. Aberrant CpG island methylation in association with transcriptional silencing has been recognized to act as an alternative to mutations and deletions to disrupt tumor suppressor gene function. A large number of genes involving fundamental cellular pathways has been shown to be affected by this epigenetic phenomenon. In this study, we investigated the possible role of CpG island hypermethylation in the transcriptional regulation of the C/EBPalpha gene in AML. In hematopoietic tumor cell lines, aberrant methylation of the C/EBPalpha promoter region was associated with transcriptional silencing. Treatment of hematopoietic cell lines, which carry a hypermethylated C/EBPalpha gene, with the demethylating agent 5-aza-2‘-deoxycytidine resulted in C/EBPalpha reexpression, as determined by real-time reverse-transcriptase polymerase chain reaction. The analysis of diagnostic bone marrow and blood specimens from adult patients with AML by methylation-specific polymerase chain reaction revealed aberrant methylation of the C/EBPalpha promoter region in 12/69 (17.4 %) samples. Hypermethylation of C/EBPalpha in AML could be detected in all cytogenetic risk groups, but was restricted to the French-American-British (FAB) subtypes M1, M2, M4 and M5. There was a trend towards a better overall survival in AML cases with C/EBPalpha hypermethylation. These data indicate that hypermethylation of the transcription factor C/EBPalpha is a common epigenetic event in adult AML. Hypermethylation-associated silencing of C/EBPalpha may, in addition to genetic aberrations, interfere with the cellular differentiation process and thus contribute to the malignant phenotype. The exploration of our growing knowledge about epigenetic aberrations in leukemogenesis may help develop novel strategies in diagnosis and treatment of AML for the future.

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ANTAGONISTIC FUNCTION OF S100 PROTEINS REGULATES AP-1 DEPENDENT GENE EXPRESSION DURING TUMOR DEVELOPMENT Christoffer Gebhardt,1,4 Michiel van der Sanden,1 Lars Hummerich,2 Kai Breuhahn,3 Julia Németh,1 Meinhard Hahn,2 Alexander Enk,4 Gerhard Fürstenberger,5 Cornelia Mauch,6 Peter Schirmacher,3 Peter Lichter,2 Peter Angel1 and Jochen Hess1

1Division of Signal Transduction and Growth Control; 2Division of Molecular Genetics; 5Division of Eicosanoids and Tumor Development, Deutsches Krebsforschungszentrum, Heidelberg, Germany; 3Department of Pathology; 4Department of Dermatology, University Hospital Heidelberg, 6Department of Dermatology, University of Cologne, Germany Email: c.gebhardt@dkfz.de Despite compelling data demonstrating a direct link between altered expression of S100 proteins located on human chromosome 1q21 and common epithelial malignancies, the knowledge of their function and mode of action in epithelial cells and in the evolution or progression of cancer is largely unknown. Here, we have identified a novel signaling pathway in epithelial cells initiated by extracellular S100A8/A9 heterodimers resulting in the activation of AP-1-dependent gene expression. Importantly, co-expression of S100A3 inhibits S100A8/A9 mediated AP-1 activation, which is in line with repression of this gene during chemically induced skin carcinogenesis in mice suggesting a negative role for S100A3 in epithelial malignancy. We found elevated levels of MMP2 and MMP9, two well-known AP-1 regulated genes, and identified S100A6 as an additional target gene of S100A8/A9 signaling in epithelial cells. Moreover, significant co-expression of S100A8 and S100A9 in concert with phosphorylation of c-Jun and elevated levels of S100A6 protein was evident in cutaneous squamous cell carcinomas of patients. Altogether, our data suggest that targeting the net activity of S100 induced signaling represents an auspicious strategy for innovative cancer prevention and/or therapy.

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Stem Cell-Based Gene Therapy for Malignant Glioma Hrvoje Miletic1, Yvonne Fischer2, Tsanan Giroglou2, Sara Litwak3, Sandra Winkeler4, Uwe Himmelreich5, Werner Stenzel1, Martina Deckert1, Andreas H. Jacobs4, Harald Neumann3, and Dorothee von Laer2 1 Abteilung für Neuropathologie, Universität zu Köln, Joseph-Stelzmann-Str. 9, D-50931 Köln, Germany 2 Georg-Speyer-Haus, Paul-Ehrlich-Strasse 42-44, D-60596 Frankfurt am Main, Germany 3Neural Regeneration Unit, Institute of Reconstructive Neurobiology, University Bonn LIFE & BRAIN Center and Hertie Foundation, Sigmund-Freud-Str. 25, 53127 Bonn, Germany. 4 Labor für Gentherapie und Molekulares Imaging, Max-Planck-Institut für Neurologische Forschung, Universität zu Köln, Gleuelerstr. 50, D-50931 Köln, Germany 5 Labor für Kernspintomographie, Max-Planck-Institut für Neurologische Forschung, Universität zu Köln, Gleuelerstr. 50, D-50931 Köln, Germany Malignant gliomas still have a dismal prognosis despite advances in neurosurgical resection and adjuvant radio- and chemotherapy. One major reason is their infiltrative growth pattern with single tumor cells migrating distantly from the solid tumor mass and infiltrating normal brain tissue. These cells are responsible for recurrent tumors near the resection cavity or even far away from it and should therefore be the main target cell population for new therapeutic interventions. One very promising therapeutic approach is the intratumoral delivery of stem cells that express therapeutic genes. Neural and mesenchymal stem cells are promising candidates for therapy of malignant glioma as they have the ability to migrate into these tumors and even track infiltrating tumor cells. However, their clinical use is limited by a low passaging capacity that impedes large scale production of genetically modified cells. In the present study we tested bone marrow derived multipotent adult progenitor cells – termed here bone marrow derived tumor infiltrating cells (BM-TIC) - that can be passaged over 100 population doublings for gene therapy of rat 9L glioma. Upon injection into the tumor/ vicinity of the tumor, BM-TIC infiltrated 9L tumors in solid as well as border areas, similar to the neural stem cell line C17.2. BM-TIC, genetically modified to express the thymidine kinase of the Herpes simplex virus (BM-TIC-tk-GFP), mediated a significant therapeutic effect with 67% of longtime survivors in vivo.

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The nuclear architecture of chromosome 17 in ductal breast carcinoma cells in comparison to non-neoplastic ductal epithelium F. Riede (1), S. Stein (2), M. Schuricke (2), J. Schwarz-Finsterle (2), C. Cremer (2), A. Walch (3), M. Werner (1), M. Hausmann (1,2) 1 = Institute of Pathology, University Hospital Freiburg 2 = Kirchhoff-Institute of Physics, University of Heidelberg 3 = Institute of Pathology, GSF Research Center, Munich Only very little is known about the nuclear architecture of tissue cells within their natural histological context and the modifications of the nuclear architecture during neoplastic degeneration. Here, we show an approach based on multi-colour Fluorescence in situ Hybridization (FISH) and quantitative 3D-fluorescence microscopic imaging that describes parameters of the spatial organization of genes and centromeres in cell nuclei in histological sections of archival tissues. Centromeric (CEP#17) and locus-specific (HER2neu) probes were hybridized to tissue microarrays prepared from formalin-fixed, paraffin-wax embedded blocks of matched pairs (n=5) of neoplastic and non-neoplastic breast epithelium. 3D-data stacks were acquired and the FISH labelled cell nuclei were interactively segmented according to the DNA counterstain. For topological analysis, a computer program was developed that allows the evaluation of many cell nuclei according to different parameters. Here, a set of six parameters was chosen: relative radial distributions of the centres of labelling intensity, normalised distances between the labelled sites and to the nuclear centre, absolute distances between the labelled sites and to the nuclear centre, nuclear centre angles, numbers of objects, and nuclear radii. The results were summarized in histograms and statistically evaluated for four different groups: (a) all five cases of non-neoplastic ductal epithelium (“normal” vs. “normal”), (b) all five cases of breast cancer (“tumour” vs. “tumour”), (c) neoplastic in comparison to non-neoplastic epithelium for each case (“tumour” vs. “normal”), (d) all cases together (“tumour” vs. “normal”). In general, matching all cases the tumours cells significantly differed from non-neoplastic cells. Centromeres 17 and the HER2neu genes appear to be localised closer to the nuclear center in tumour cell nuclei than in nuclei of non-neoplastic epithelium although the nuclear radius of the tumour cells increased and the number of centromeres and genes has been amplified in some cases. Comparing non-neoplastic cells between the cases separately reveals no significant differences of the location of centromere 17 and HER2neu among the individuals. However, each case shows individual differences between non-neoplastic cells and tumour cells. These results indicate that the nuclear architecture in normal epithelium may be conserved, independently from the individual, whereas the nuclear architecture in tumour cells may be subjected to strong individual differences. Therefore further investigations of the nuclear architecture during cancerogenesis may be necessary.

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β-catenin regulates hTERT (human telomerase RT-component) expression arguing for a tumor stem cell population in colorectal carcinomas Falk Hlubek, Elke Hiendlmeyer1, Thomas Brabletz1, Angela Haynl, Hermann Herbst2, Thomas Kirchner, Andreas Jung Pathologisches Institut, LMU-München; 1Pathologisches Institut, Universität Erlangen-Nürnberg; 2Gerhard-Domagk-Institut für Pathologie, Universität Münster Introduction: Tumor stem cells have been shown to be the biologically relevant fraction of tumor cells in leukemias, mamma carcinomas, and glioblastomas. The canonical Wnt signaling pathway with the central transcriptional activator protein β-catenin has been shown to be an important regulator of stem cells. Therefore, we intended to characterize the potential fraction of tumor stem cells in human colorectal adenocarcinomas (hCRC) on the basis of their nuclear expression of β-catenin and hTERT (human telomerase RT component) which are both markers for adult stem cells. Experimental Procedures: The localisation of β-catenin and hTERT was investigated using immunohistochemistry (IHC) of hCRC applying specific monoclonal antibodies for β-catenin or hTERT respectively. Binding of β-catenin or TCF-4 to the promoter enhancer of the hTERT gene was analysed using Electric Mobility Shift Assays (EMSA) and chromatin immunoprecipitations (ChIP). The β-catenin/TCF-4 driven transcriptional activation of the hTERT gene was investigated applying luciferase-reporter assays and β-catenin knock-down experiments using siRNA as functional tests. Data: Nuclear co-expression of β-catenin and hTERT was found in tumor cells located in the zone of the invasion front of 25 well differentiated hCRC using IHC specific for β-catenin and hTERT. Moreover, the expression of hTERT is regulated by β-catenin/TCF-4 on the level of transcription. The hTERT promoter/enhancer harbors four consensus binding sites for TCF-4 (TBE) which are specifically bound by recombinant TCF-4 or endogenous β-catenin-complex using EMSAs or ChIPs respectively. Additionally, the transcription of the hTERT gene depends on β-catenin/TCF-4 as mutating the TBEs of the hTERT promoter/enhancer results in a lowered activity of the hTERT promoter/enhancer using luciferase-reporter assays. Moreover, a reduction in the hTERT expression was induced in colorectal tumor cell lines by β-catenin knock-down experiments applying β-catenin specific siRNA. Conclusions: Tumor stem cells might exist also in hCRC. They resemble a tiny fraction of the whole tumors and are located at their invasion fronts. The potential tumor stem cells are characterized by the nuclear expression of β-catenin and hTERT. Moreover, β-catenin drives the expression of hTERT supporting the role of β-catenin as an inductor of stemness in intestinal cells. The functional testing of the stemness of this fraction of cells has now to be shown.

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Extracellular KIT receptor mutants, commonly found in core binding factor AML, are constitutively active and respond to the kinase inhibitors imatinib mesylate and SU5416. S. Horn1, J. Cammenga1, U. Bergholz1, G. Sommer2, P. Besmer2, W. Fiedler3, and C. Stocking1

1Molecular Pathology Group at the Heinrich-Pette-Institut, Hamburg, Germany; 2Developmental Biology Program, Sloan-Kettering Institute, New York; 3Dept. of Hematology and Oncology, Universitätsklinikum Hamburg-Eppendorf, Hamburg, Germany. Activating mutations of the receptor tyrosine kinase KIT have been linked to several hematologic neoplasias, including acute myelogenous leukemia (AML) and mastocytosis. Mutations in the extracellular domain of the KIT receptor are almost exclusively found in AML patients carrying fusions affecting members of the core binding factor (CBF) gene family and correlate with a high risk of relapse. We demonstrate that these complex insertion and deletion mutations lead to constitutive activation of the KIT receptor, which induces factor-independent growth of IL3-dependent cells. Mutation of the evolutionary conserved amino acid D419 within the extracellular domain was sufficient to constitutively activate the KIT receptor, although high expression levels were required. Dose-dependent growth inhibition and apoptosis were observed using either the protein tyrosine kinase inhibitor imatinib mesylate or SU5416. Blocking the PI3K-AKT pathway, in contrast to the MEK-MAPK pathway, was sufficient to inhibit proliferation and to induce apoptosis. Our data show that the addition of kinase inhibitors to conventional chemotherapy might be a new therapeutic option for CBF-AML expressing mutant KIT.

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Oncogenic KRAS Synergizes with the Tumor Suppressor Apc in Intestinal Tumor Progression Klaus-Peter Janssen1, Paola Alberici2, Mechthild Wagner3, Sabrina Carpentier4, Fatima El Marjou5, Wilhelm Ansorge3, Daniel Louvard4, Riccardo Fodde2, and Sylvie Robine4. 1Dept. of Surgery, Klinikum rechts der Isar, TUM, Munich, Germany 2Erasmus University Medical Center, Rotterdam, The Netherlands3European Molecular Biology Laboratory, Heidelberg, Germany 4UMR144-Institut Curie Paris, France The process of colon carcinogenesis involves the progressive disruption of cellular signalling cascades which regulate cell proliferation, cell death and differentiation. The Ras signaling pathway is known to play a pivotal role in these processes. However, its precise role in cancer initiation and progression remains unclear. Therefore, we have established a transgenic mouse model that expresses oncogenic KRAS-V12G in intestinal epithelia under control of the murine villin promoter. The expression of oncogenic Ras induced spontaneous tumorigenesis in the transgenic animals, unexpectedly even in the absence of Apc mutations. By crossing the KRAS-V12G transgenic mice with Apc1638N mice, which carry a mutated allele of the tumor suppressor gene Apc, we have obtained a complementary model that mimics later stages of the human disease. The resulting animals demonstrated a drastic synergistic effect on tumor initiation and progression. A ten-fold increase in tumor number,as well as strongly increased mortality and morbidity were observed. It has been reported that oncogenic Ras promotes beta-catenin phosphorylation at tyrosine residues, leading to its release from E-cadherin. The increase in cytoplasmatic beta-catenin, and its translocation to the nucleus lead to Wnt activation. Preliminary evidence suggests that this enhanced Wnt-signalling expands the putative tumor stem cell compartment, thus contributing to the drastic increase in tumorigenesis in the compound model. In order to further elucidate the specific contribution of oncogenic KRAS to tumorigenesis, we compared the expression profiles of the KRASV12G and Apc

+/1638N/KRASV12G tumors, revealing surprising differences between both groups.

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Mesenchymal stem cells (MSCs) and TRAIL-induced apoptosis in prostate tumours Kallifatidis, G1., Beckermann, B1., Wenger, T.1, Schubert, M.2, Krause, U.2, Su, Dr.3, Friess, H.3, Büchler, P.3 and Herr, I.1 1Molecular Urooncology, DKFZ, Heidelberg, Germany, 2Policlinic and 3Department of Surgery, University of Heidelberg, Germany Aims: The death ligand tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a potent inducer of apoptosis in transformed cells, while sparing most normal cell types. Homing of manipulated mesenchymal stem cells (MSCs) to glioma cells or of embryonal progenitor cells to lung metastases in mouse xenograft models has been demonstrated in recent reports. Also, endothelial progenitor cells are known for homing into tumours and participation in formation of new tumour blood vessels. MSCs may contribute to formation of tumour stroma and tumour blood vessels and thus be ideal vehicles for the targeted transfer of therapeutic genes. Thus, lentiviral transduction of MSCs with TRAIL may result in TRAIL-overexpressing tumour stroma and endothelial cells which in turn induce programmed cell death in surrounding prostate tumour cells. Methods: TRAIL sensitivity of established and freshly isolated human prostate carcinoma cells towards recombinant TRAIL was tested by MTT assays and annexin-staining followed by flow cytometry. For therapeutic intervention, lentiviral vectors are constructed for expression of TRAIL. MSCs are selected from intraoperatively harvested bone marrow and tumour-specific migration to prostate tumour cell lines was detected in vitro and to prostate tumour xenografts on nude mice in vivo. For in vivo detection, MSCs are marked by lentiviral GFP. Transdifferentiation to tumour stroma and endothelial-like cells upon homing in tumours is analyzed by surface staining. Involvement of MSCs in paracrine induction of apoptosis by TRAIL in tumour cells is examined in vitro by three-dimensional tumour reconstructs and in vivo by measurement of tumour growth and staining of cryosections. Results: Tumour-specific in vitro homing of non-manipulated MSCs selected from bone marrow of patients was found. Lentiviral GFP-control vector transduced to MSCs resulted in a transfection efficiency of 20%. To increase transfection efficiency we inserted a puromycin expression cassette into the lentiviral vector to enable selection and thereby enrichement of transduced MSCs. Injection of lentiviral GFP-expressing MSCs into the tail vein of nude mice xenografted with human subcutaneously growing prostate tumours demonstrated in vivo homing to the tumour cells. Our actual work focuses on transdifferentiation of non-manipulated and lentiviral transduced MSCs, induction of paracrine apoptosis in tumour cells by TRAIL expressing MSCs as well as in derived stromal and endothelial cells and finally, prevention of tumour growth. Conclusions: Autologous MSCs may be a powerful tool for transfer of therapeutic genes into tumours, e.g. for specific inhibition of tumour apoptosis.

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Biocomputing of chromosomal aberrations G. Kretha*, S. Pazhanisamya, J. v. Hasea, N. Keppera , C. Cremera,c,d aKirchhoff Institute for Physics, University Heidelberg, Germany cInterdisciplinary Center for Scientific Computing, University Heidelberg d The Jackson Laboratory, Bar Harbor/ME The spatial organization of the genome in the interphase nucleus has far reaching consequences for radiation biophysics. For example, the existence of chromosome territories and their spatial distribution in the human cell nucleus is expected to pose serious constraints on the induction of specific chromosome aberrations by ionizing radiation. Experimentally, using Fluorescence in situ hybridization (FISH) chromosome painting, confocal laser scanning microscopy, and digital image analysis, a non-random, non-statistical positioning of chromosome territories (CTs) in human lymphocyte cell nuclei has been observed for certain CTs. This distribution has been maintained throughout primate evolution, suggesting a functional significance. Such a clearly non random organization of nuclear genome “macro” structure has raised the question to what extend this organization favor specific interchange aberration frequencies. The spatial proximity of certain CTs or even of clusters of CTs have to increase the respective exchange yields significantly in comparison to a random association of CTs. Computer simulations of nuclear genome structure based on experimentally known general features allow to predict chromosome aberration yields induced by ionizing radiation in interphase nuclei on the single cell level. As an example, in this report computer simulated arrangements of CTs in human cell nuclei models (“1-Mbp Spherical Chromatin Domain Model”) were assumed to calculate interchange frequencies between all heterologous CT pairs. For the positioning of CTs in the virtual nuclear volume, both a statistical and a gene density correlated arrangement was assumed. Under the assumption of an initial repair rate, the calculated absolute aberration frequencies were found to be in good agreement with an experimental study of Arsuaga et al. 2004.

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Establishment and characterization of long-term glioblastoma stem cell cultures Nils-Ole Schmidt, Hauke Günther, Hildegard Meissner, Manfred Westphal, Katrin LamszusDept. of Neurosurgery, University Medical Center Eppendorf, Hamburg, Germany Glioblastomas contain a small subpopulation of cells that display a stem stem cell-like phenotype. Serum-free culture conditions with appropriate growth factors can support the survival of neural stem cells and were recently shown to also support the growth of glioblastoma stem cells (e.g. Galli et al., Cancer Res 2004). We used established protocols to generate permanent glioblastoma stem cell lines from tumors of 8 different glioblastoma patients. All 8 cell lines have now been grown for at least 12 passages, and for up to 25 passages (over 1.5 yrs in culture), and are stably expandable. Most cell lines grow entirely as neurospheres, however, 3 cultures grow partly adherent. In all cultures expression of the neural stem cell markers nestin, CD133, musashi-1, SOX2 and Bmi-1 was detected using immunocytochemistry and/or RT-PCR, and most cells co-expressed GFAP. Under differentiating conditions, expression of the neuronal and oligodendroglial markers MAP-2 and GalC, respectively, was inducible, whereas nestin expression was lost by most cells. More than 50% of the cells were clonogenic and formed new secondary spheres in limiting dilution assays. Upon intracerebral injection into nude mice, the cells formed diffusely invasive tumors. To conclude, long-term propagation of glioblastoma stem cells as permanent cell lines is feasible. These cells can be used to develop specifically tailored therapies, and to test anti-invasive strategies in a highly invasive murine model.

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Isolation and differentiation of mesenchymal stem cells from lipoaspirates Lückerath K., Matthey M., Rutkowski A., Stoeckius M., Maiworm A., Popova S., Holzhauer C., Spriestersbach A., Roitzheim B., Pansky A., Tobiasch E. University of Applied Sciences Bonn-Rhein-Sieg, Von-Liebig-Str. 20, 53359 Rheinbach Embryonic stem cells are always associated with ethical issues. In addition, the high plasticity of those cells poses the threat of unwanted proliferation, which may lead to cancer development. Adult stem cells, in contrast, have a limited differentiation capacity, thus a lower carcinogenic potential. They are commonly derived from bone marrow, umbilical cord and peripheral blood. However, the isolation procedures are cost and time intensive, the cell yield is low and the material is usually obtained from unhealthy donors. Lipoaspirate from plastic surgeries was found to be an alternative source of mesenchymal stem cells. Its advantage lies in its unrestricted availability in large amounts from healthy donors. This study aimed at isolating these stem cells by a simple and inexpensive method. The lipoaspirate separated into an upper-fat-tissue- and a bottom-fluid-layer, both containing stem cells. The bottom layer contained large amounts of erythrocytes which interfered with cell adhesion. Four procedures were compared with regard to the yield of cells capable of multilineage differentiation. They differed in the layer used and the application of erythrocyte lysis. The highest cell number was obtained when erythrocytes were lysed in the bottom layer pellet. The isolated cells were assessed by FACS-analysis. Their differentiation capacity towards the osteogenic-, myogenic- and adipogenic lineage was determined. Specific staining methods and RT-PCR were used for evaluation. Our study showed that adipose tissue is a valuable source for adult mesenchymal stem cells. These cells are easy and inexpensive to harvest in adequate amounts. This is in clear contrast to stem cells derived from other sources. Moreover, a standardisation of the isolation method will allow the direct comparison of different scientific approaches. This will contribute to the development of future therapeutic applications without ethical concerns, thus circumventing the use of embryonic stem cells.

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SASH1: A NEW CANDIDATE TUMOR SUPPRESSOR IN COLORECTAL CANCER

Melanie Martini, Caroline Rimkus, Daniela Mayr, Bernhard Holzmann and Klaus-Peter Janssen Department of Surgery, Klinikum rechts der Isar, TUM, Munich, Germany SASH1, a member of the recently described SLY-family of SH3/SAM adapter proteins, is a new potential tumor suppressor gene in colorectal cancer. SASH1 has already been implicated in the tumorigenesis of breast and other solid cancers. SASH1 is normally widely expressed in human and murine tissues, with the exception of immune cells. We have observed a strong downregulation of SASH1 expression in human colorectal tumors. This downregulation was clearly correlated with poor prognosis for patient survival. In addition, we have detected LOH (loss of heterozygosity) of the SASH1 locus in 15 percent of the analyzed murine digestive tumors (n=20). However, the cellular processes and molecular pathways in which SASH1 is involved are still unknown. To address this question in vivo, we are currently generating a conditional and tissue specific SASH1 knock-out mouse. Moreover, we have characterized SASH1 in vitro by expressing human SASH1 transiently or stably in various epithelial cell lines. An overexpression significantly reduced cell proliferation. Immunofluorescence microscopy revealed co-localisation of SASH1 with the cortical actin cytoskeleton in lamellipodia, membrane ruffles and microspikes. This implies that the new tumor suppressor SASH1 might play a role in the control of the actin cytoskeleton, responsible for tumor cell migration and invasion. Upon loss of function of SASH1, tumor cells could acquire higher migratory and invasive properties.

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Allelic silencing at 13q14.3: a novel oncogenic mechanism Daniel Mertens1, Stephan Wolf1, Cora Mund2, Dirk Kienle 3, Sibylle Ohl1, Petra Schroeter1, Frank Lyko2, Hartmut Döhner3, Stephan Stilgenbauer3, Peter Lichter1

1) Department of Molecular Genetics 2) Department of Epigenetics, DKFZ, Heidelberg, Germany; 3) Department of Internal Medicine III, University of Ulm, Germany INTRODUCTION: Genomic material from chromosomal band 13q14.3 distal to RB1 is recurrently lost in a variety of human neoplasms, yet lack of point mutations in candidate tumor suppressor genes and downregulation of these genes in tumors indicate a pathomechanism of epigenetic nature localized in the critical region. METHODS: Candidate tumor suppressor genes are down regulated by more than a factor of two in tumors with loss of one copy of the critical region. In addition, the presence of large non-coding RNA genes in 13q14.3 is reminiscent of imprinted regions where only one gene copy is active. Therefore we tested candidate tumor suppressor genes for monoallelic expression in healthy probands using single nucleotide polymorphisms and sequencing of RT-PCR products. Genotyping parents of these probands allowed allocation of the parental origin of either gene copy. In addition, we performed FISH experiments to measure replication timing of the two copies of the critical region to find out whether they are functionally different. As transcriptional activity and replication timing are effectuated by chromatin packaging, we used combined bisulfite-restriction (COBRA) analyses and bisulfite sequencing to assess DNA methylation of the critical region. Treatment of cultured cells with inhibitors of DNA-methyltransferases and histone-deacetylases allowed functional correlation of chromatin modification with expression of candidate tumor suppressor genes localized in the critical region. RESULTS: In line with an imprinting mechanism, we find that the two copies of the critical region replicate asynchronously, suggesting differential chromatin packaging of the two copies of 13q14.3. In addition, we could detect monoallelic silencing of genes localized in the critical region and expression of one gene copy only. However, expression originated from either the maternal or paternal copy, excluding an imprinting mechanism. DNA methylation analyses showed one of the CpG islands of the region to be methylated. Demethylation of DNA and histone hyperacetylation induced biallelic expression, while replication timing was not affected. CONCLUSIONS: We propose that differential replication timing represents an early epigenetic mark that distinguishes the two copies of 13q14.3, resulting in differential chromatin packaging and monoallelic expression. Accordingly, deletion of the single active copy of the tumor suppressor encoding region at 13q14.3 results in significant down regulation of the candidate genes and loss of function, providing a model for the interaction of genetic lesions and epigenetic silencing at 13q14.3.

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Organotypic Cultures: A Model for Human Interfollicular Epidermal Stem Cell Niches Sonja Muffler, Hans-Jürgen Stark, Mara Amoros and Petra Boukamp Deutsches Krebsforschungszentrum, Heidelberg, Germany Epidermal stem cells (ESCs) are located in hair follicles and interfollicular epidermis and are surrounded by a specific microenvironment - the stem cell niche. The factors regulating this niche are not known. However, several markers are proposed to define ESCs and their location. To investigate whether organotypic cultures (OTCs) are suitable in vitro models for interfollicular ESCs, we compared the distribution of proposed ECS markers in skin and two different OTCs: OTCs with a dermal equivalent consisting of a type I collagen gel and newly developed long-term OTCs with a scaffold-stabilized dermal equivalent, both with integrated fibroblasts. By immunofluorescence we analyzed the distribution of β1 and α6 integrins, keratin 15 and 19, p63 and melanoma chondroitin sulphate proteoglycan (MCSP). While the marker distribution in OTCs was generally comparable to skin, scaffold-OTCs were clearly superior to collagen-OTCs, particularly obvious for β1 and α6 integrins. However, both integrins, K15 and p63 were expressed by all basal cells. Only MCSP and K19 were restricted to individual cells making them best candidates for further stem cell characterization. Collectively, our results suggest that stem cell niches re-establish in OTCs and that OTCs provide a promising model for human interfollicular ESCs under controlled in vitro conditions.

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Identification of putative cancer stem cells in pancreatic adenocarcinoma cell lines Monika Olempska, Patricia Alice Eisenach, Hendrik Ungefroren, Heiner Schäfer, Holger Kalthoff Klinik für Allgemeine Chirurgie und Thoraxchirurgie, Sektion Molekulare Onkologie Pancreatic adenocarcinoma represents the fourth leading cause of cancer-associated mortality in industrialized countries and is among the most difficult to treat effectively. Clearly, new approaches for successful therapy are urgently needed. Growing evidence suggests that for various types of cancer growth and therapeutic failure are contingent on cancer stem cells. Thus far, the presence of cancer stem cells in pancreatic cancer has not been demonstrated. Here, we investigated whether pancreatic cancer cell lines that have been established and maintained for many years in culture contain a subpopulation of putative stem cells. We initially quantified the mRNA expression levels of two stem cell markers, BCRP1 (breast cancer-resistant protein 1) and isoform 2 of CD133 (CD133-2) and showed that all six pancreatic cancer cell lines tested express significantly higher levels of BCRP1 than non-malignant cells or two other malignant non-pancreatic cell lines. Elevated CD133-2 expression was found in five out of six pancreatic cancer cell lines tested. Using flow cytometry analysis we were able to confirm the presence of BCRP1 on the cell surface in all six lines. CD133 surface expression however was detectable only in one cell line. In addition, we detected putative stem cells by taking advantage of the ability of BCRP1-expressing cells to extrude the fluorescent dye Hoechst 33342. Our data suggest that established pancreatic cancer cell lines contain a subpopulation of putative stem cells that may be a promising target for therapeutic drug research.

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Migration of B16-F1 melanoma cells in the neural crest pathways after transplantation into the neural tube of the chick embryo

Oppitz M, Busch C, Schriek G, Just L, Metzger M, Drews U Institute of Anatomy, Department of Experimental Embryology, University of Tübingen, Österbergstr. 3, 72074 Tübingen, Germany Melanocytes originate from the neural crest. In the basal layer of the epidermis the melanocyte precursor cells form an adult stem cell population from which skin melanomas may arise. We asked whether the migratory behaviour during invasive growth of melanoma cells was the same as during embryonic neural crest cell migration. To test the hypothesis we transplanted mouse melanoma B16-F1 cells transfected with VASP-GFP construct into the neural tube of the 2 day chick embryo. Migration of melanoma cells was followed with live fluorescence microscopy and characterised in serial paraffin sections by immunohistochemistry with HMB-45 as marker for melanoma cells, the neural crest cell specific antibody HNK-1, and by in situ hybridization for mouse specific repetitive genomic sequence mL1. Apoptosis of melanoma cells was assessed with anti-caspase 3 immunohistochemistry and TUNEL staining. The migration pattern of B16-F1 cells was compared with the pattern of chick neural crest cells in whole mounts stained with HNK-1.

12 h after injection of a cell suspension into the trunk neural tube the melanoma cells formed clusters at the levels of the intersegmental clefts between somites. 2 days after injection melanoma cells were identified in the medial neural crest pathway, in particular in the sympathetic trunk ganglia. 4 days after transplantation melanoma cells were found among the chick melanocyte precursor cells above the neural tube (“staging area”). In the periphery of the embryo only some apoptotic mouse melanoma cells were encountered. While melanoma cells injected in the neural tube finally underwent apoptosis, melanoma cells transplanted in the eye cup formed melanomas.

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Stroma-induced in vitro differentiation of human AML cells Kallal Pramanik1, Axel Greiner2, Albrecht M. Müller1 1Institut für Medizinische Strahlenkunde und Zellforschung,Universität Würzburg. Germany. 2Pathologisches Institut, Universität Würzburg. Germany The embryonic micro-environment can alter the phenotype and control the tumorgenicity of transplanted tumour cells. Earlier we reported the appearance of markers of erythroid lineage differentiation in human acute myeloid leukaemia (AML) cells following microinjection into murine blastocysts (Dürr et al., 2003, Oncogene). This finding suggests that the embryonic environment can regulate the proliferation and differentiation of leukaemic cells. In the present study we have analyzed the consequences of stromal environment-induced changes in human AML cells upon in vitro co-culture of AML and stromal cell lines in terms of differentiation and proliferation properties. For this purpose we co-cultured established human AML cell lines on mitotically inactivated stromal cells derived from different murine embryonic and foetal haematopoietic sites such as yolk sac, Aorta-Gonad-Mesonephros (AGM) region and foetal liver. To score for co-culture-induced changes in the AML cells we compared the morphology, histo-chemical properties, immuno-phenotype, proliferation rate and gene expression in cocultured and non-cocultured control cells. Our results show that, upon 6 days of co-culture with an AGM and Foetal liver derived but not with many other stromal cells, human AML Kasumi-1 cells showed an evident decrease in proliferation and colony formation efficiency together with the appearance of differentiated morphologies. Direct co-culturing of Kasumi-1 cells resulted in the up-regulation of myelo-monocytic lineage cell surface markers. Direct coculturing has proved to be more efficient in reducing the proliferation rate then the indirectly cocultured Kasumi-1 cells. Moreover, the proliferation rate of the cocultivated cells can be rescued upon removal of the feeder layer. Gene-expression analysis showed a significant decrease in the transcription of the AML1-ETO fusion gene. In summary, we have developed an in vitro reversible system, capable of reducing proliferation and inducing differentiation in human AML cells. Further it will be important to decipher the cellular mechanism by which stromal environment is capable of regulating tumour cell proliferation and differentiation.

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Hypermethylation and Transcriptional Downregulation of the CITED4 Gene at 1p34.2 in Oligodendroglial Tumors with Allelic Losses on 1p and 19q Bjoern Tews, Peter Roerig, Christian Hartmann, Meinhard Hahn, Jörg Felsberg, Britta Blaschke, Michael Sabel, Annegret Kunitz, Grischa Toedt, Kai Neben, Axel Benner, Andreas von Deimling, Guido Reifenberger and Peter Lichter Institut für Neuropathologie, Düsseldorf Loss of heterozygosity (LOH) on chromosomal arms 1p and 19q is frequent in oligodendroglial tumors and associated with sensitivity to radio- and chemotherapy as well as favorable prognosis. Using microarray-based expression profiling we found that oligodendroglial tumors with LOH 1p/19q showed significantly lower expression of the CBP/p300-interacting transactivator with glutamic acid/aspartic acid-rich carboxyl-terminal domain 4 gene (CITED4) at 1p34.2 as compared to gliomas without LOH 1p/19q. Mutational analysis showed no mutations in the CITED4 coding sequence. However, LOH 1p/19q and low expression of CITED4 transcripts were closely associated with CITED4 promoter hypermethylation. Treatment of CITED4 hypermethylated glioma cells with 5-aza- 2’-deoxycytidine and trichostatine A markedly increased CITED4 expression. Furthermore, CITED4 hypermethylation is a significant predictor of longer survival in patients with oligodendroglial tumors. Taken together, our results suggest CITED4 as a putative tumor suppressor gene that is epigenetically silenced in oligodendroglial tumors with LOH 1p/19q and represents a powerful prognostic marker.

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DISSECTING THE WNT-PATHWAY AND ITS COOPERATION WITH ONCOGENIC KRAS IN COLORECTAL TUMOR PROGRESSION Franziska Rohde1, Matthias Ernst2, Sylvie Robine3, Bernhard Holzmann1, and Klaus-Peter Janssen1 1Dept. of Surgery, Klinikum rechts der Isar, TUM, Munich, Germany 2Ludwig Institute for Cancer Research, Melbourne, Australia 3UMR144-Institut Curie, Paris, France Wnt signaling is involved in the regulation of stem cell renewal and cellular differentiation. Moreover, the Wnt signaling pathway is misregulated in the majority of colon cancers. The concomitant activation of Wnt signaling, due to either APC loss of function or activating mutations in CTNNB1, and of the KRAS oncogene defines a subset of patients with advanced tumors and poor prognosis. However, this synergism is not well understood on the molecular level. In order to dissect the cooperation between the Wnt and the KRAS pathways, we have generated several murine models: Firstly, we have generated a mutant mouse that combines expression of oncogenic Kras-V12G in intestinal epithelia with a mutation at the Apc locus (Apc1638N). This compound line showed a drastic increase in tumorigenesis. Secondly, we combined the pVillin-Kras-V12G mice with a strain expressing a N-terminally truncated, oncogenic version of beta-catenin (A33∆N-βcat) in intestinal epithelia. This allows us to distinguish between downstream effects of Apc that are caused by activation of beta-catenin, or that are independent of beta-catenin. The A33∆N-βcat mice develop colonic precursor lesions (ACF) but show only occasionally progression towards tumor formation. Interestingly, we found that only a specific subset of canonical Wnt targets is differentially regulated in A33∆N-βcat mice. We are currently analyzing the pVillin-Kras-V12G/ A33∆N-βcat compound animals on the biochemical, molecular and histopathological level.

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Epigenetic Modifications in Hodgkin's and Anaplastic Large Cell Lymphoma Timm Schlegel+, Askar Akimzhanov+, Laszlo Krenacs*, Sergej Chuvpilo+, Alois Palmetshofer+, Edgar Serfling+ +Department of Molecular Pathology, Institute of Pathology, University of Würzburg, Würzburg, Germany *Laboratory of Tumor Pathology and Molecular Diagnostics, Institute of Biotechnology, Bay Zoltan Foundation for Applied Research, Szeged, Hungary Hodgkin and Anaplastic Large Cell Lymphoma (ALCL) are two distinct, but probably related malignancies that share some common features, e.g. expression of the surface marker CD30 (the "Ki-1" antigen). It has been shown recently that the loss of the immunoglobulin receptor repertoire, including the B cell receptor in Hodgkin's lymphoma, and the T cell receptor in ALCL, respectively, as well as a variety of Ig-associated signalling molecules, is characteristic for both entities. Both the B cell and the T cell signalling cascade would, under non-pathological conditions, eventually lead to the activation of several transcripion factors such as NFATc1. Expression studies in patient's material as well as surrogate cell lines have revealed, that this particular transcription factor is also not expressed in these cells. It could be demonstrated that this is due to epigenetic silencing by promotor hypermethylation, as revealed by the bisulfite DNA sequencing technique. Additionally, re-expression of this transcription factor, as well as BOB.1 and PLCgamma2 could be successfully evoked by treatment with the demethylating agent 5'-Aza-2'-deoxycytidine. As CpG-Methylation has also been shown for other promotors, it is likely that aberrant hypermethylation plays a major role in the pathogenesis of these lymphomas.

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Enhanced neurogenesis of adult stem cells using inhibitors of histone deacetylases Siebzehnrübl FA, Buslei R, Eyupoglu IY, Seufert S, Hahnen E and Blümcke I. Dept Neuropathology, University Erlangen Directed differentiation of neural stem cells will be of paramount importance for reconstructive therapy strategies in central nervous system diseases. Epigenetic regulatory mechanisms such as histone (de-)acetylation are likely to influence stem cell fate decision during normal development and aging. Here, we investigated the effect of histone deacetylase inhibition on the differentiation of adult neural stem cells. We isolated subventricular zone stem cells from postnatal day six Wistar rats and cultured them as neurospheres. Following differentiation we treated neurosphere-derived cells with different histone deacetylase inhibitors (HDACI) and subsequently determined differentiated cell types using immunofluorescence microscopy. The second generation HDACI suberoylanilide hydroxamic acid (SAHA), MS-275 and M344 were able to increase the population of �III tubulin positive neurons up to six-fold compared to mock-treated controls. NeuroD, CyclinD2 and Btg3 were identified as target genes for neuronal differentiation. We further investigated the propensity of HDACI to alter the differentiation into various subtypes of neurons, i.e. GABAergic, dopaminergic or gluatamatergic.

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A novel solid phase PCR for quantitative methylation analysis

Sievers S*°, Götze S*, Kuhnen C°, Müller O* *Max-Planck-Institut für molekulare Physiologie 44227 Dortmund, Germany °Institut für Pathologie der Ruhr-Universität Bochum an den BG-Kliniken Bergmannsheil 44789 Bochum, Germany Abstract: Epigenetic silencing of genes by hypermethylation of promoter sequences constitutes an important step in tumourigenesis and has equally been described as first or second hit. The hypermethylation of the Adenomatous Polyposis Coli (APC) promoter is a common event in a variety of tumours, for example in colon cancer or lung cancer. The APC protein is not only a key player in the wnt signalling pathway which when turned on leads to cell proliferation but exhibits diverse functions in the maintenance of chromosomal stability, cytoskeleton regulation and DNA binding. In the majority of tumours, methylated as well as non-methylated alleles can be detected by standard methods implying either monoallelic or inconsistent methylation of cancer cells or the presence of non-tumour cells in the sample. Therefore a quantitative analysis of methylation should be helpful in evaluating the role of promoter hypermethylation in cancer formation. Standard methylation-sensitive (ms) PCR assays are not quantitative. Methods for quantitative analysis of methylation which include ms-real-time PCR or ms-SNuPE (Single Nucleotide Primer Extension) are laborious and costly. A novel ms solid phase PCR to test APC promoter 1A methylation was developed which combines the amplification on a solid phase with subsequent colorimetric detection in an ELISA based format. This method promises to be low-cost and also suitable for medium-throughput. In a first PCR reaction a fragment of the APC promoter is amplified in a non-methylation specific manner thereby preventing PCR bias. The second ms solid phase PCR is carried out with an immobilized non-methylation specific primer and a biotinylated ms primer. After extensive washing the PCR product can be detected via a streptavidin-biotin horseradish peroxidase complex. The applicability for quantitative analysis was tested by applying mixtures of different percentages of methylated and non-methylated DNA. Thereby a calibration curve was set up. The novel ms solid phase PCR promises a fast, accurate and low-cost quantitative analysis of methylation levels in promoters of tumour suppressor genes.

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Transcription-independent activities of a transcription factor: lessons learnt from p53 Daniel Speidel and Wolfgang Deppert HEINRICH-PETTE-INSTITUT FÜR EXPERIMENTELLE VIROLOGIE UND IMMUNOLOGIE, D-20251 HAMBURG, GERMANY The tumor suppressor p53 is a central mediator in the cellular response to genotoxic stress and pivotal in preventing growth of potentially tumorigenic cells. Primarily established as a transcription factor, p53 induces expression of a plethora of target genes that mediate its anti-proliferative effects. In addition, recent evidence has suggested transcription-independent mechanisms as an important mode of p53 activity. p53 impairs proliferation by induction of three different stress response programs: cell cycle arrest, senescence, or apoptosis. While arrest of cell cycle and senescence keep a damaged cell alive, induction of the apoptotic program efficiently leads to its elimination. Although many aspects of p53 function are very well understood, which signals determine selectivity of the p53 response and thus cause p53 to either rescue or eliminate a damaged cell remains unclear.

In order to dissect the complex network of p53 signaling, we have analyzed the integration of the diverse p53 activities in mouse fibroblasts and human tumor-derived cell lines. We treated cells with different irradiation schemes and thereby specifically induced different outcomes of a stress response. Based on a comparative approach we have identified determinants that initiate an anti- or a pro-apoptotic p53 response within the context of p53-independent stress signaling. We show that the primary, transcription-mediated p53 response in these cells is anti-apoptotic, whereas induction of p53-mediated apoptosis requires an additional non-transcriptional p53 activity provided by high intracellular levels of activated p53. High p53 levels selectively accumulated in the cytosol of cells following distinct apoptosis-inducing stimuli, leading to activation of the pro-apoptotic protein Bax and apoptosis. In agreement with these results, the anti-apoptotic p53 response in severely damaged cells could be converted to apoptosis by artificially increasing intracellular p53 levels. Our results demonstrate the impact and in vivo relevance of non-transcriptional mechanisms for p53 function. Achievement of a selective response by the cooperative action of transcriptional and non-transcriptional activities is most likely not a unique feature of p53 regulation but might be a more general principle utilized by other transcription factors.

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Epigenetic silencing of the candidate tumor suppressor gene PROX1 in sporadic breast cancer Beatrix Versmold 1, Jörg Felsberg 2, Denise Ehrentraut 2, Juliane Köhler 1, Juergen A. Hampl 3, Gabriele Röhn 3, Dieter Niederacher 4, Beate Betz 4, Torsten Pietsch 2, Rita K. Schmutzler 1,5 and Andreas Waha 2,5,6 (1) = Division of Molecular Gyneco-Oncology, Department of Gynecology and Obstetrics, and Center of Molecular Medicine Cologne (CMMC), University of Cologne, Germany (2) = Department of Neuropathology, University of Bonn Medical Center, Bonn, Germany (3) = Department of Neurosurgery, University of Cologne, Germany (4) = Department of Gynecology and Obstetrics, University of Düsseldorf, Germany Changes of DNA methylation play a central role in carcinogenesis. Extensive hypermethylation and consecutive transcriptional silencing of tumorsuppressor genes have been documented in multiple tumor entities including breast cancer. In a microarray based genome-wide methylation analysis of three sporadic breast carcinomas we identified a CpG island within the first intron of the PROX1 (prospero related gene 1) gene that showed hypermethylation compared to normal breast tissue. Lack of PROX1 transcription has been associated with down-regulation of the cell-cycle inhibitors CDKN1B and CDKN1C and abnormal cellular proliferation. Moreover, somatic mutations and epigenetic silencing of PROX1 has recently been demonstrated in hematologic malignancies. We, therefore, proceeded to investigate CpG island methylation of PROX1 in a series of 9 primary breast cancer samples by bisulfite sequencing. Seven out of 9 (78%) tumor samples showed a significant accumulation of methylated CpG sites along with a significant reduction of PROX1 transcription compared to normal breast tissues of the same patients. Additionally treatment of breast cancer cell lines with demethylating agents showed reactivation of PROX1 transcription. In an expanded set of 35 breast cancer samples a total of 29 (83%) showed a reduced transcription of PROX1. Secondary we determined PROX1 RNA-expression in a set of 38 brain metastases of primary breast carcinomas and detected a significantly reduced expression compared to normal breast tissue (p<0.001) and primary breast carcinomas (p<0.05), respectively. In summary, we have identified PROX1 as a novel target gene that is hypermethylated and transcriptionally silenced in primary and metastatic breast cancer. In line with recent findings on PROX1 inactivation in lymphomas, our data suggest that inactivation of this homeobox gene might also play a pivotal role in malignant transformation and progression of breast cancer and that epigenetic silencing seems to be the major mode of its inactivation.

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Stem cell markers in testicular seminoma Isabella Gashaw1, Ralph Brehm2, Rüdiger Behr1,3, Oliver Dushaj1, Rainer Grobholz4, Kurt Werner Schmid5, Christian von Ostau6, Katharina Biermann7, Martin Bergmann2, Elke Winterhager1

1 Institute of Anatomy, University Hospital Essen, 45122 Essen, 2 Institute of Veterinary Anatomy, University of Giessen, Giessen 3 German Primate Center, Göttingen, 4 Department of Pathology, Ruprecht-Karls University Heidelberg, Mannheim, 5 Institute of Pathology, University Hospital Essen, 6 Department of Urology, University Hospital Essen 7 Institute of Pathology, University of Bonn, Bonn, Pure seminoma develops from a primordial germ cell and express genes characteristic for stem cells such as KIT (c-kit), SCGF, OCT4 or NANOG. As found in microarrays and by RT-PCR analyses, we confirmed an enhanced expression of these genes in 40 seminoma specimens considering progressing tumor stages (pT1, pT2, pT3). In addition, we identified for the first time other stem cell genes in seminoma: PROM1, BOB1 and MCFD2. Prominin 1 (PROM1) has been originally identified on neuroepithelial stem cells in mice and is a pentaspan transmembrane glycoprotein. The mRNA was weekly expressed in normal testicular tissue and significant enhanced in seminoma cells. This high expression persists in all tumor stages and the protein has been localised to seminoma cells by immunohistochemistry. BOB1 (POU2AF1) represents a transcriptional factor expressed in human hematopoietic stem cells. We could localise the BOB1 protein to nuclei of spermatogonia cells in normal testes and the transcript was highly upregulated in seminoma tissues. The protein has been immunolocalised in seminoma cells whereas the infiltrating leucocytes were negative for BOB1. Like the expression pattern of the stem cell growth factor SCGF, the newly identified MCFD2 (NCGF) gene encoding for a neuronal stem cell factor has been localised to Sertoli cells of normal testes. However, the mRNA was still highly expressed in the germ cell tumor and the protein could be detected in pure seminoma cells of all tumor tissues. MCFD2 was present in gonocytes from second trimester of gestation, but absent in third trimester, indicating a role of this protein in fetal germ cell development. In conclusion, the constitutively upregulation of stem cell markers in seminoma confirm the model that stem cell genes -not any more present in germ cells of adult testis- are maintained in seminoma cells pointing to the fact that the origin of this tumor is stem cell and not germ cell derived.

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