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In the majority of human tumors the oncogenic transcription factor c-MYC is deregulated and contributes to the formation of many biologically important tumor properties. These include the induction of cell cycle progression, transformation, genomic instability and immortalization. So far it was unclear which target genes of c-MYC mediate the effects. Using genome-wide approaches we identified a large number of c-MYC target genes. Subsequently, we characterized some target genes for their role in c-MYC-induced genomic instability and immortalization. The protein deacetylase SIRT1 was found to be an important mediator of c-MYC-induced immortalization. Using in situ analyses of colorectal cancer specimens we demonstrated that c-MYC is a regulator of the identified target genes in human tumors thus implicating their relevance for tumorigenesis in humans.

Die zunehmende Alterung unserer Gesellschaft hat zur Folge, dass altersbedingte Erkrankungen, wie z. B. Typ-2 Diabetes, vermehrt auftreten. Dies bringt sowohl wirtschaftliche als auch große soziale Probleme mit sich. Ernährungsbezogene Präventionsansätze befassen sich daher u. a. mit dem Einfluss von Nahrungsbestandteilen auf die Expression krankheitsrelevanter Gene. Die Deacetylase SIRT1, die durch kleine Moleküle in ihrer Aktivität reguliert werden kann, steuert diverse Stoffwechselprozesse, die mit altersbedingten Erkrankungen in Verbindung stehen. In der Vergangenheit wurden verschiedene Screeningmethoden zur Detektion SIRT1-modulierender Substanzen entwickelt. Allerdings weisen viele der verfügbaren SIRT1-Screeningmethoden Nachteile auf, etwa die Generierung von Artefakten aufgrund von Fluoreszenzdetektion und/oder -markierung. Um dieses Problem zu lösen wurde in der vorliegenden Arbeit eine MALDI-TOF MS-basierte Screeningmethode entwickelt. Anhand dieser Methode kann die Deacetylierung eines Peptidsubstrates aufgrund seiner um 42 Da verringerten Masse detektiert werden, ohne dass eine Substratmarkierung erforderlich ist. Die Methode wurde so optimiert, um sie einerseits als Hochdurchsatz-Screeningmethode, und andererseits zur nachfolgenden Charakterisierung der ermittelten SIRT1-Modulatoren verwenden zu können. Die Methodenentwicklung sowie das Screening einer Naturstoffbibliothek wurden mit einem unmarkierten und mit einem fluoreszenzmarkierten Peptid durchgeführt. Das unmarkierte Peptid diente dabei der Erzielung artefaktfreier Ergebnisse. Das fluoreszenzmarkierte Peptid wurde hingegen zur Detektion artifizieller SIRT1-Aktivatoren eingesetzt, um strukturelle Ähnlichkeiten zu anderen SIRT1-Aktivatoren aufzeigen zu können. Aus dem Screening resultierten acht SIRT1-Inhibitoren und ein artifizieller SIRT1-Aktivator. Diese Substanzen wurden in vitro und in verschiedenen Zellsystemen überprüft. Dabei zeigte der stärkste SIRT1-Inhibitor NP1 im Zellsystem eine reduzierte p53-Deacetylierung. Der artifizielle SIRT1-Aktivator NP9, der mit einem IC50 -Wert von 1,7 µM zudem ein sehr starker p300-Inhibitor ist, wies eine stark antiinflammatorische Wirkung auf. Anhand der Histonacetyltransferase p300 wurde gezeigt, dass die MALDI-TOF MS Methode sich auch zur Detektion der Umkehrreaktion eignet. Zudem bietet die Methode die Möglichkeit, die Aktivität vieler weiterer posttranslationaler Enzyme zu messen., The progressive increase in life expectancies of our society results in an increasing prevalence of age-related diseases like type-2 diabetes. This causes economic as well as vast social problems. Therefore, nutrition related preventive approaches include for example the influence of dietary components on the expression of disease related genes. The deacetylase SIRT1 can be regulated by small molecules and controls several metabolic processes that influence age-related diseases. Several methods to screen for SIRT1 modulating compounds have been developed in the past. However, many of the available SIRT1 screening methods bear disadvantages such as artifact generation because of fluorescence detection and/or labeling. Hence, in the present work a more advantageous MALDI-TOF MS based screening method was developed. Applying this method, deacetylation of a peptide substrate can be detected by a 42 Da mass shift without the need of any substrate labeling. The method was optimized to serve as a high-throughput screening system as well as for subsequent characterization of detected SIRT1 modulators. Method development as well as screening of a natural product library was conducted using an unlabeled and a fluorescently labeled peptide. The unlabeled peptide was used to generate artifact-free results. In contrast, the fluorescently labeled peptide was applied for the detection of artificial SIRT1 activators, in order to demonstrate structural similarities to other SIRT1 activating compounds. The screening resulted in eight SIRT1 inhibitors and one artificial SIRT1 activator. These compounds were further tested in vitro and in different cell lines. Cell treatment with the most potent SIRT1 inhibitor NP1 revealed reduced p53 deacetylation. The artificial SIRT1 activator compound NP9, which is also a very strong inhibitor of p300 with an IC50 value of 1.7 µM, showed a strong antiinflammatory effect. By employing the histone acetyltransferase p300, the applicability of the MALDI-TOF MS based method for the reverse reaction was demonstrated. Moreover, the presented method provides the opportunity to measure the activity of many other post-translationally active enzymes.

Atherosclerosis is a chronic inflammatory disease that is based on the interaction between inflammatory cell subsets and specific cells in the arterial wall. SIRT1 deacetylates histone and non-histone proteins and has been implicated in protective effects of caloric restriction on lifespan and metabolic pathways in yeast, nematodes, and mice. In the vasculature of rodents, SIRT1 mediates vasodilatation through the release of endothelial nitric oxide synthase-derived nitric oxide and scavenges reactive oxygen species. Using a genetic loss-of-function approach, SIRT1 has been shown to interfere with crucial steps of endothelial activation and atherogenesis by suppressing NFκB signaling: Partial SIRT1 deletion in ApoE-/- mice prevented expression of endothelial adhesion molecules thereby hampering the extravasation of circulating monocytes. In monocyte-derived macrophages SIRT1 deletion reduced the expression of the scavenger receptor lectin-like oxidized low-density lipoprotein receptor 1 (Lox-1) resulting in reduced foam cell formation and atherosclerosis. Moreover, it was reported that SIRT1 regulates the activity of liver X-receptor, thereby promoting ABCA1-driven reverse cholesterol transport in plaque-resident macrophages slowing foam cell formation. Finally, SIRT1 suppressed the expression of endothelial tissue factor, and thus exerted anti-thrombotic properties during induced carotid thrombosis in mice. These findings indicate protective effects of SIRT1 in atherogenesis and thrombosis at an experimental level and highlight the opportunity to translate this concept from bench to bedside. Indeed, SIRT1 activators are available and have been shown to exert beneficial effects at the preclinical level in obesity and type 2 diabetes mellitus (T2DM). SIRT1 activators are currently being evaluated in phase II clinical trials in patients with T2DM. The concept of SIRT1 activation appears a promising strategy for novel therapeutic approaches in patients with atherothrombosis.

The growing number of patients with type 2 diabetes stimulates the development of new antidiabetic drugs. New targets are addressed, as SGLT2 in the kidney or enzymes of glucose metabolism in the liver which might help to lower glucose levels and to overcome insulin resistance. The forthcoming years will show which of the drugs in clinical trials will get marketing authorization.

Vielleicht kann – wie Neil Diamond einst meinte – Rotwein uber eine verlorene Liebe hinweg helfen; pharmazeutisch interessanter ist, ob wirklich Rotwein und hier vor allem das Resveratrol fur das “French Paradoxon” verantwortlich ist. Demnach schutzen sich die Franzosen trotz vielen Tabakonsums und cholesterinreicher Ernahrung mit Rotwein vor Herzinfarkten.