3rd field of activity
Prof. Dr. Boris Schmidt (TU Darmstadt, Organische Chemie)
Never-in-mitosis A related kinase 1 (NEK1) is a dual serine-threonine and tyrosine kinase which is involved in ciliogenesis, DNA damage response and apoptosis. NEK1 phosphorylates voltage-dependent anion channel 1 (VDAC1) and stabilizes the mitochondrial membrane potential, which is important to prevent apoptosis. Additionally NEK1 expression is up-regulated upon chemical or radiative induction of DNA double strand breaks. Recently the group of Prof. Löbrich (also member of Grk1657) revealed the role of NEK1 in DNA double strand break repair by homologous recombination. It regulates phosphorylation of Rad54 dependent on the cell cycle to either ensure replication fork stability or permit homologous recombination.
Inhibition of NEK1 may facilitate the investigation of the role of NEK1 in DNA damage response and apoptosis as well as treatment for cancer. A decrease in activity of NEK1 in cancer cells leads to unrepaired DSB and cell death eventually.
High levels of NEK1 are present in renal cell carcinoma, which in turn possess a high resistance to genotoxic treatment. The combination of irradiation or a genotoxic agent and a NEK1 inhibitor may overcome this therapeutic resistance.
The overall aim of this project is to find new scaffolds for inhibitors by in silico screening of established scaffolds, to investigate their structure-activity-relationship (SAR) and eventually to develop a highly selective and active small molecule inhibitor against the NEK1 kinase domain by medicinal chemistry approaches. The developed structures shall be profiled in an in vitro kinase assay and in vivo by cellular Rad54 phosphorylation and zebra fish toxicity assay. The project therefore aims to be highly interdisciplinary and applicants should be able to work in a highly interdisciplinary field.
PD Dr. Bodo Laube (TU Darmstadt, Biologie)
Glioblastoma multiforme (GBM) is one of the most common and aggressive malignant primary brain tumors in humans characterized by a high radio-resistance and a high degree of invasive growth. GBMs secrete the excitatory neurotransmitter glutamate at concentrations sufficient i) to stimulate proliferation, infiltration, and cell survival of the tumor cells and ii) to induce excitotoxic neuronal cell death in the surrounding tissue. The apoptotic cell death of neurons as well as the promoting effect of these high glutamate concentrations on tumor cells depends on the spatial and temporal stimulation of different Ca2+-permeable ionotropic glutamate-receptors of the NMDAR-subtype and the subsequent activation of distinct signal transduction pathways. Under physiological conditions NMDAR-mediated activation of the cAMP-responsive element binding transcriptionfactor (CREB) by glutamate is crucial for brain development and neuronal plasticity involved in learning and memory formation. In the first phase of the GrK-funding we found in glioblastoma cells i) that glutamate improved the DNA damage response (DDR) of ionizing radiation- (IR) induced DNA double-strand breaks (DSBs) and ii) that blocking NMDAR-mediated glutamatergic signaling resulted in a decreased cell survival and a sensitization to IR. In addition we could show that both glutamate and IR activate the CREB pathway and that the DDR is inhibited by CREB antagonists. Remarkably, even activation of the NMDAR on its own resulted in DSBs in glioblastoma cells, similar to those found upon IR with doses of 0.5 Gy. A recent publication in Cell (1) reports that NMDAR-stimulation triggers the formation of type II topoisomerase-mediated DSBs in the promoters of early-response genes that is crucial for experience-driven synaptic changes associated with learning and memory. Furthermore, several data suggest that IR can induce gene expression by radiation-responsive promotor-sequences. Thus, in the next phase of the GrK we will analyze in a comparative study the expression profile of distinct genes upon NMDAR- and IR-induced DSBs to get insights in the mechanisms and the signal pathways involved in the specific induction of DSBs in promoter regions facilitating the expression of early-response genes.
Dr. Florian Frohns (TU Darmstadt, Biologie)
Accurate detection and repair of DNA damage occurring after genotoxic stress are preconditions for the maintenance of genomic integrity of cells. For the repair of DNA double-strand breaks (DSBs), two main pathways exist, non-homologous end-joining (NHEJ), and homologous recombination (HR). The latter one is error free since it operates during S and G2 phase when the sister chromatid is available as a template for repair. We recently discovered a mechanism that regulates the activity of Rad54, a protein involved in HR: In response to DNA damage Rad54 becomes activated through its phosphorylation by the protein kinase Nek1 in late G2, but not in S phase . This project now is designed to study this mechanism in an in vivo situation using different mice strains with mutations in either Rad54 or Nek1. Based on our longstanding experience with studies of the irradiation response in vivo [2, 3] we designed an experimental approach that allows us to analyze the regulation and the contribution of different repair pathways to the DNA damage repair in different mouse tissues in a cell cycle specific manner. In this project, major emphasis will be placed on the analysis of selected developmental stages of the central nervous system. Beside the effects of a missing or de-regulated HR on DNA repair, also the consequences on cellular survival and cell cycle checkpoint induction will be analyzed.
Prof. Dr. Barbara Drossel (TU Darmstadt, Physik)
This theoretical project shall model the decision processes in cells following ionizing radiation by constructing the relevant network of interactions (phosphorylation, binding, gene expression, etc) and investigating it by computer simulations. Initially, the focus shall be on the dependence of p21 activation on p53 and the cell cycle stage. Later, contextual information, such as signals from tumor nekrosis factor alpha, transforming growth factor beta, and chromatin structure shall be included. All investigations shall be performed in close collaboration with experimental groups.