Zur Bedeutung der polyTC-Motive in den Promotoren und 5‘-UTR-Bereichen konstitutiv exprimierter Pflanzengene

Das Gen der V-ATPase UE c1 aus der Zuckerrübe (Beta vulgaris) besitzt im Promotor und der 5’UTR je eine Polypyrimidinbox. Solche Motive aus sich häufenden TC-Alternierungen sind in pflanzlichen Promotoren und 5’UTRs ein häufig auftretendes Motiv mit bislang weitgehend ungeklärter Funktion. Am Beispiel des Promotors Y11037 des konstitutiv exprimierten V-ATPase c1 Gens wurden in dieser Arbeit gezeigt, dass die enthaltenen Polypyrimidin-Boxen ein funktional notwendiger Bestandteil des c1-Promotors sind. Die c1-Promotoraktivität fiel nach der Mutation der Polypyrimidin-Box1 (Box1 5’seitig der TATA-Box) um 95%, beziehungsweise nach Mutation der Polypyrimidin-Box2 (Box2 Position +44 – +63 in 5’UTR) um 37%. Weitere Versuche zeigten jedoch, dass allein die Existenz einer Polypyrimidin-Box nahe der TATA-Box nicht hinreichend ist um eine verstärkte Promotoraktivität zu gewährleisten. Es konnten aus Arabidopsis thaliana homologe Proteine des Polypyrimidin-bindenden Transkriptionsfaktors BBR aus der Gerste (Hordeum vulgare, Santi 2003) und Gbp aus der Sojabohne (Glycine max, Sangwan 2002) kloniert und aufgereinigt werden. Diese AtBR-Proteine mit einer stark homologen, jedoch bislang unbekannten DNA-, beziehungsweise Polypyrimidin-bindenden Domäne (Sangwan 2002) sind Vertreter einer gemeinsamen Familie von Transkriptionsaktivatoren. Für die AtBR-Transkriptionsfaktoren wurden zunächst die Voraussetzungen für eine spezifische Interaktion mit den Polypyrimidin-Boxen des c1-Promotors belegt. Eine subzelluläre Lokalisation eines N-terminalen GFP-AtBR1-Fusionsproteins ergab eine spezifische Akkumulation im Zellkern transient transformierter Beta vulgaris Zellsuspensionskultur. Zusätzlich konnte durch ein Cobombardment eines c1-Promotor-LUC-Konstrukts mit AtBR-Expressionsvektoren eine Aktivitätserhöhung des c1-Promotors in vivo festgestellt werden. Mittels Gelretentionsanalyse (EMSA) wurden in vitro für zwei AtBR-Transkriptionsfaktoren (AtBR1 und AtBR4) eine spezifische Interaktion mit den Polypyrimidin-Boxen des c1-Promotors nachgewiesen, wobei die Polypyrimidin-Box1 eine mindestens zweifach größere Affinität zu den AtBR-Transkriptionsfaktoren besitzt als die Polypyrimidin-Box2. Dieser Befund legt im Zusammenhang mit dem drastischen Aktivitätsverlust dach der Inaktivierung der Polypyrimidin-Box1 eine, in Abhängigkeit von der Bindeaffinität der AtBR-Transkriptionsfaktoren mit den Polypyrimidin-Motiven vorliegende Promotoraktivität (Transkriptionseffektivität) nahe. Weiterhin ergab ein in Arabidopsis-Pflanzen durchgeführtes Gen-Silencing für die AtBR-Transkriptionsfaktoren AtBR1, AtBR2 und AtBR3 erste Evidenzen für eine AtBR beeinflusste c1-Promotoraktivität. Alle Ergebnisse implizieren, dass die Frage nach der funktionalen Bedeutung von Polypyrimidin-Boxen und deren Rolle als Bindemotiv in den Promotoren und 5’UTRs sich durch eine Interaktion von Vertretern der Proteinfamilie der AtBR-Transriptionsfaktoren erklärt. Der wirtschaftliche Einsatz des c1-Promotors oder allgemein von Promotorsequenzen mit Polypyrimidin-Motiven zur Expression von Nutzgenen in transgenen Pflanzen muss die Zusammenhänge durch die Wechselwirkung von AtBR-Transkriptionsfaktoren für den gezielten Einsatz berücksichtigen. Weitere Grundlagenforschung, insbesondere die Suche nach mit AtBR interagierenden Proteinen und die Bedeutung der einzelnen AtBR-Isoformen, sind für ein tieferes Verständnis unerlässlich

Establishment of a patient-derived orthotopic osteosarcoma mouse model

Background: Osteosarcoma (OS) is the most common pediatric primary malignant bone tumor. As the prognosis for patients following standard treatment did not improve for almost three decades, functional preclinical models that closely reflect important clinical cancer characteristics are urgently needed to develop and evaluate new treatment strategies. The objective of this study was to establish an orthotopic xenotransplanted mouse model using patient-derived tumor tissue. Methods: Fresh tumor tissue from an adolescent female patient with osteosarcoma after relapse was surgically xenografted into the right tibia of 6 immunodeficient BALB/c Nu/Nu mice as well as cultured into medium. Tumor growth was serially assessed by palpation and with magnetic resonance imaging (MRI). In parallel, a primary cell line of the same tumor was established. Histology and high-resolution array-based comparative genomic hybridization (aCGH) were used to investigate both phenotypic and genotypic characteristics of different passages of human xenografts and the cell line compared to the tissue of origin. Results: A primary OS cell line and a primary patient-derived orthotopic xenotranplanted mouse model were established. MRI analyses and histopathology demonstrated an identical architecture in the primary tumor and in the xenografts. Array-CGH analyses of the cell line and all xenografts showed highly comparable patterns of genomic progression. So far, three further primary patient-derived orthotopic xenotranplanted mouse models could be established. Conclusion: We report the first orthotopic OS mouse model generated by transplantation of tumor fragments directly harvested from the patient. This model represents the morphologic and genomic identity of the primary tumor and provides a preclinical platform to evaluate new treatment strategies in OS

NASH limits anti-tumour surveillance in immunotherapy-treated HCC.

Hepatocellular carcinoma (HCC) can have viral or non-viral causes1-5. Non-alcoholic steatohepatitis (NASH) is an important driver of HCC. Immunotherapy has been approved for treating HCC, but biomarker-based stratification of patients for optimal response to therapy is an unmet need6,7. Here we report the progressive accumulation of exhausted, unconventionally activated CD8+PD1+ T cells in NASH-affected livers. In preclinical models of NASH-induced HCC, therapeutic immunotherapy targeted at programmed death-1 (PD1) expanded activated CD8+PD1+ T cells within tumours but did not lead to tumour regression, which indicates that tumour immune surveillance was impaired. When given prophylactically, anti-PD1 treatment led to an increase in the incidence of NASH-HCC and in the number and size of tumour nodules, which correlated with increased hepatic CD8+PD1+CXCR6+, TOX+, and TNF+ T cells. The increase in HCC triggered by anti-PD1 treatment was prevented by depletion of CD8+ T cells or TNF neutralization, suggesting that CD8+ T cells help to induce NASH-HCC, rather than invigorating or executing immune surveillance. We found similar phenotypic and functional profiles in hepatic CD8+PD1+ T cells from humans with NAFLD or NASH. A meta-analysis of three randomized phase III clinical trials that tested inhibitors of PDL1 (programmed death-ligand 1) or PD1 in more than 1,600 patients with advanced HCC revealed that immune therapy did not improve survival in patients with non-viral HCC. In two additional cohorts, patients with NASH-driven HCC who received anti-PD1 or anti-PDL1 treatment showed reduced overall survival compared to patients with other aetiologies. Collectively, these data show that non-viral HCC, and particularly NASH-HCC, might be less responsive to immunotherapy, probably owing to NASH-related aberrant T cell activation causing tissue damage that leads to impaired immune surveillance. Our data provide a rationale for stratification of patients with HCC according to underlying aetiology in studies of immunotherapy as a primary or adjuvant treatment

Longitudinal MRI study after carbon ion and photon irradiation: shorter latency time for myelopathy is not associated with differential morphological changes

Background!#!Radiation-induced myelopathy is a severe and irreversible complication that occurs after a long symptom-free latency time if the spinal cord was exposed to a significant irradiation dose during tumor treatment. As carbon ions are increasingly investigated for tumor treatment in clinical trials, their effect on normal tissue needs further investigation to assure safety of patient treatments. Magnetic resonance imaging (MRI)-visible morphological alterations could serve as predictive markers for medicinal interventions to avoid severe side effects. Thus, MRI-visible morphological alterations in the rat spinal cord after high dose photon and carbon ion irradiation and their latency times were investigated.!##!Methods!#!Rats whose spinal cords were irradiated with iso-effective high photon (n = 8) or carbon ion (n = 8) doses as well as sham-treated control animals (n = 6) underwent frequent MRI measurements until they developed radiation-induced myelopathy (paresis II). MR images were analyzed for morphological alterations and animals were regularly tested for neurological deficits. In addition, histological analysis was performed of animals suffering from paresis II compared to controls.!##!Results!#!For both beam modalities, first morphological alterations occurred outside the spinal cord (bone marrow conversion, contrast agent accumulation in the musculature ventral and dorsal to the spinal cord) followed by morphological alterations inside the spinal cord (edema, syrinx, contrast agent accumulation) and eventually neurological alterations (paresis I and II). Latency times were significantly shorter after carbon ions as compared to photon irradiation.!##!Conclusions!#!Irradiation of the rat spinal cord with photon or carbon ion doses that lead to 100% myelopathy induced a comparable fixed sequence of MRI-visible morphological alterations and neurological distortions. However, at least in the animal model used in this study, the observed MRI-visible morphological alterations in the spinal cord are not suited as predictive markers to identify animals that will develop myelopathy as the time between MRI-visible alterations and the occurrence of myelopathy is too short to intervene with protective or mitigative drugs

Postsurgical Adjuvant Tumor Therapy by Combining Anti-Angiopoietin-2 and Metronomic Chemotherapy Limits Metastatic Growth

Antiangiogenic tumor therapy has failed in the adjuvant setting. Here we show that inhibition of the Tie2 ligand angiopoietin-2 (Ang2) effectively blocks metastatic growth in preclinical mouse models of postsurgical adjuvant therapy. Ang2 antibody treatment combines well with low-dose metronomic chemotherapy (LDMC) in settings in which maximum-dose chemotherapy does not prove effective. Mechanistically, Ang2 blockade could be linked to quenching the inflammatory and angiogenic response of endothelial cells (ECs) in the metastatic niche. Reduced EC adhesion molecule and chemokine expression inhibits the recruitment of tumor-promoting CCR2(+)Tie2(-) metastasis-associated macrophages. Moreover, LDMC contributes to therapeutic efficacy by inhibiting the recruitment of protumorigenic bone marrow-derived myeloid cells. Collectively, these data provide a rationale for mechanism-guided adjuvant tumor therapies

Switching of vascular phenotypes within a murine breast cancer model induced by angiopoietin-2

Sustained growth of solid tumours can rely on both the formation of new and the co-option of existing blood vessels. Current models suggest that binding of angiopoietin-2 (Ang-2) to its endothelial Tie2 receptor prevents receptor phosphorylation, destabilizes blood vessels, and promotes vascular permeability. In contrast, binding of angiopoietin-1 (Ang-1) induces Tie2 receptor activation and supports the formation of mature blood vessels covered by pericytes. Despite the intense research to decipher the role of angiopoietins during physiological neovascularization and tumour angiogenesis, a mechanistic understanding of angiopoietin function on vascular integrity and remodelling is still incomplete. We therefore assessed the vascular morphology of two mouse mammary carcinoma xenotransplants (M6378 and M6363) which differ in their natural angiopoietin expression. M6378 displayed Ang-1 in tumour cells but no Ang-2 in tumour endothelial cells in vivo. In contrast, M6363 tumours expressed Ang-2 in the tumour vasculature, whereas no Ang-1 expression was present in tumour cells. We stably transfected M6378 mouse mammary carcinoma cells with human Ang-1 or Ang-2 and investigated the consequences on the host vasculature, including ultrastructural morphology. Interestingly, M6378/Ang-2 and M6363 tumours displayed a similar vascular morphology, with intratumoural haemorrhage and non-functional and abnormal blood vessels. Pericyte loss was prominent in these tumours and was accompanied by increased endothelial cell apoptosis. Thus, overexpression of Ang-2 converted the vascular phenotype of M6378 tumours into a phenotype similar to M6363 tumours. Our results support the hypothesis that Ang-1/Tie2 signalling is essential for vessel stabilization and endothelial cell/pericyte interaction, and suggest that Ang-2 is able to induce a switch of vascular phenotypes within tumours