Herunterregulation des Proto-Onkogens c-Jun durch das CCAAT/Enhancer bindende Protein alpha in der Myelopoese und in der akuten myeloischen Leukämie (AML)

Verschiedene Transkriptionsfaktoren spielen eine Rolle in der Entwicklung myeloischer Zellen. c-Jun gehört zur Familie der AP-1 Transkriptionsfaktoren und ist ein Koaktivator des Transkriptionsfaktors PU.1, der für die Differenzierung zu Monozyten wichtig ist. C/EBPa ist ein an den CCAAT-Enhancer bindender Transkriptionsfaktor. Wird C/EBPa exprimiert, differenzieren sich bipotente myeloische Zellen zu neutrophilen Granulozyten. Die durch den Induktor TPA hervorgerufene Differenzierung zu Monozyten wird gehemmt. C/EBPa Knockout-Mäuse entwickeln dementsprechend keine ausgereiften Granulozyten. Gleichzeitig lässt sich in der Leber dieser Mäuse ein dramatischer Anstieg der mRNA für c-Jun beobachten. Wir legen hier erste Beweise dafür vor, dass C/EBPa die Expression von c-Jun herunterreguliert, um so die Differenzierung zu Granulozyten voranzutreiben. Ektoper Anstieg der C/EBPa Expression vermindert den c-Jun mRNA Spiegel und die Aktivität des menschlichen c-Jun Promotors wird herunterreguliert. C/EBPa und c-Jun können über ihre Leucin-Zipper-Domänen interagieren. In dieser Bindung kann c-Jun nicht mehr an die DNA binden. So wird die Fähigkeit c-Juns vermindert, den eigenen Promotor durch die proximale AP-1 Box zu autoregulieren. Andererseits kann durch eine Überexpression c-Juns die durch C/EBPa induzierte Differenzierung zu Granulozyten gehemmt werden. Zudem konnten wir beobachten, dass in AML-M2 Patienten mit dominant negative C/EBPa Mutationen die c-Jun Expression höher war verglichen zu AML-M2 Patienten ohne dominant negativen C/EBPa Mutationen. Somit schlagen wir ein Modell vor, in dem C/EBPa sowohl die Expression als auch die Transaktivationskapazitiät von c-Jun herunterreguliert, um die Differenzierung von myeloischen Vorläuferzellen zu Granulozyten zu fördern

Synthesis of Structurally Diverse N-Substituted Quaternary-Carbon-Containing Small Molecules from α,α-Disubstituted Propargyl Amino Esters.

N-containing quaternary stereocenters represent important motifs in medicinal chemistry. However, due to their inherently sterically hindered nature, they remain underrepresented in small molecule screening collections. As such, the development of synthetic routes to generate small molecules that incorporate this particular feature are highly desirable. Herein, we describe the diversity-oriented synthesis (DOS) of a diverse collection of structurally distinct small molecules featuring this three-dimensional (3D) motif. The subsequent derivatisation and the stereoselective synthesis exemplified the versatility of this strategy for drug discovery and library enrichment. Chemoinformatic analysis revealed the enhanced sp3 character of the target library and demonstrated that it represents an attractive collection of biologically diverse small molecules with high scaffold diversity.ERC (FP7/2007-2013; 279337/DOS) Marie Curie Fellowship (2013-IEF-626191) AstraZeneca IDB Cambridge International Scholarshi

Mitochondrial oxidative stress and nitrate tolerance – comparison of nitroglycerin and pentaerithrityl tetranitrate in Mn-SOD(+/- )mice

BACKGROUND: Chronic therapy with nitroglycerin (GTN) results in a rapid development of nitrate tolerance which is associated with an increased production of reactive oxygen species (ROS). According to recent studies, mitochondrial ROS formation and oxidative inactivation of the organic nitrate bioactivating enzyme mitochondrial aldehyde dehydrogenase (ALDH-2) play an important role for the development of nitrate and cross-tolerance. METHODS: Tolerance was induced by infusion of wild type (WT) and heterozygous manganese superoxide dismutase mice (Mn-SOD(+/-)) with ethanolic solution of GTN (12.5 μg/min/kg for 4 d). For comparison, the tolerance-free pentaerithrityl tetranitrate (PETN, 17.5 μg/min/kg for 4 d) was infused in DMSO. Vascular reactivity was measured by isometric tension studies of isolated aortic rings. ROS formation and aldehyde dehydrogenase (ALDH-2) activity was measured in isolated heart mitochondria. RESULTS: Chronic GTN infusion lead to impaired vascular responses to GTN and acetylcholine (ACh), increased the ROS formation in mitochondria and decreased ALDH-2 activity in Mn-SOD(+/- )mice. In contrast, PETN infusion did not increase mitochondrial ROS formation, did not decrease ALDH-2 activity and accordingly did not lead to tolerance and cross-tolerance in Mn-SOD(+/- )mice. PETN but not GTN increased heme oxygenase-1 mRNA in EA.hy 926 cells and bilirubin efficiently scavenged GTN-derived ROS. CONCLUSION: Chronic GTN infusion stimulates mitochondrial ROS production which is an important mechanism leading to tolerance and cross-tolerance. The tetranitrate PETN is devoid of mitochondrial oxidative stress induction and according to the present animal study as well as numerous previous clinical studies can be used without limitations due to tolerance and cross-tolerance

Senescent fibroblast-derived Chemerin promotes squamous cell carcinoma migration

Aging is associated with a rising incidence of cutaneous squamous cell carcinoma (cSCC), an aggressive skin cancer with the potential for local invasion and metastasis. Acquisition of a senescence-associated secretory phenotype (SASP) in dermal fibroblasts has been postulated to promote skin cancer progression in elderly individuals. The underlying molecular mechanisms are largely unexplored. We show that Chemerin, a previously unreported SASP factor released from senescent human dermal fibroblasts, promotes cSCC cell migration, a key feature driving tumor progression. Whereas the Chemerin abundance is downregulated in malignant cSCC cells, increased Chemerin transcripts and protein concentrations are detected in replicative senescent fibroblasts in vitro and in the fibroblast of skin sections from old donors, indicating that a Chemerin gradient is built up in the dermis of elderly. Using Transwell® migration assays, we show that Chemerin enhances the chemotaxis of different cSCC cell lines. Notably, the Chemerin receptor CCRL2 is remarkably upregulated in cSCC cell lines and human patient biopsies. Silencing Chemerin in senescent fibroblasts or the CCRL2 and GPR1 receptors in the SCL-1 cSCC cell line abrogates the Chemerin-mediated chemotaxis. Chemerin triggers the MAPK cascade via JNK and ERK1 activation, whereby the inhibition impairs the SASP- or Chemerin-mediated cSCC cell migration.Taken together, we uncover a key role for Chemerin, as a major factor in the secretome of senescent fibroblasts, promoting cSCC cell migration and possibly progression, relaying its signals through CCRL2 and GPR1 receptors with subsequent MAPK activation. These findings might have implications for targeted therapeutic interventions in elderly patients

Manganese superoxide dismutase and aldehyde dehydrogenase deficiency increase mitochondrial oxidative stress and aggravate age-dependent vascular dysfunction

AimsImbalance between pro- and antioxidant species (e.g. during aging) plays a crucial role for vascular function and is associated with oxidative gene regulation and modification. Vascular aging is associated with progressive deterioration of vascular homeostasis leading to reduced relaxation, hypertrophy, and a higher risk of thrombotic events. These effects can be explained by a reduction in free bioavailable nitric oxide that is inactivated by an age-dependent increase in superoxide formation. In the present study, mitochondria as a source of reactive oxygen species (ROS) and the contribution of manganese superoxide dismutase (MnSOD, SOD-2) and aldehyde dehydrogenase (ALDH-2) were investigated.Methods and resultsAge-dependent effects on vascular function were determined in aortas of C57/Bl6 wild-type (WT), ALDH-22/2, MnSOD+/+, and MnSOD+/ mice by isometric tension measurements in organ chambers. Mitochondrial ROS formation was measured by luminol (L-012)-enhanced chemiluminescence and 2-hydroxyethidium formation with an HPLC-based assay in isolatedheart mitochondria. ROS-mediated mitochondrial DNA (mtDNA) damage was detected by a novel and modified version of the fluorescent-detection alkaline DNA unwinding (FADU) assay. Endothelial dysfunction was observed in aged C57/Bl6 WT mice in parallel to increased mitochondrial ROS formation and oxidative mtDNA damage. In contrast, middle-aged ALDH-22/2 mice showed a marked vascular dysfunction that was similar in old ALDH-22/2 mice suggesting that ALDH-2 exerts agedependent vasoprotective effects. Aged MnSOD+/2 mice showed the most pronounced phenotype such as severely impaired vasorelaxation, highest levels of mitochondrial ROS formation and mtDNA damage.ConclusionThe correlation between mtROS formation and acetylcholine-dependent relaxation revealed that mitochondrial radical formation significantly contributes to age-dependent endothelial dysfunction