Prof. Dr. H.-J. Hauser
Gene regulation and differentiation; GBF, Braunschweig
Research focus and projects:
1. Tumor suppressive activity of IRF-1 (Kröger/Hauser)
The interferon regulatory factor-1 (IRF-1) is one of ten known members of the IRF family. IRF-1 is a nuclear transcriptional activator. It is expressed constitutively at a very low level in almost every cell type. IRF-1 shows tumor suppressive activity under certain conditions and IRF-1 overexpression inhibits cell growth. We showed IRF-1 mediated growth inhibition of diverse tumorigenic cells. A fusion protein in which IRF-1 was fused to the hormon binding domain of the human estrogen receptor (hER) rendering activation of IRF-1 function dependent on -estradiol treatment. -esradiol mediated IRF-1 activation leads to strong proliferation inhibition of various transformed cells expressing this fusion protein. Expression of the IRF-1 fusion protein in cells transformed by the expression of the two oncogenes c-myc and c-Ha-ras leads to growth inhibition, induction of the immune modulators IFN- and MHC class I proteins, and to the reversion of the transformed phenotype. Subcutanous injection of these cells into nude mice prevents tumor growth after IRF-1 activation. Injection of IRF-1 expressing tumor cells in immune competent mice also abolishes tumor growth and leads to the activation of the adaptive immune system. T-cell mediated immunity directed against respective tumor antigens from the primary inoculated cells results in a vaccination for wild type tumors. We have show that IRF-1 suppresses tumor growth through both.: 1) a direct antitumor growth effect and 2) enhanced immune cell recognition of the tumor cells. To establish a preclinical proof of IRF-1 as an vaccine for tumor therapy, it is suggested to investigate IRF-1 effects on proliferation, transformation and tumor growth in human tumor cells. To test the IRF-1 activity in a broad spectrum of tumor types human tumor cells from different origins like breast cancer cells, hepatocellular carcinoma cells and cervix carcinoma cells should be used. First, stable cells clones expressing the activatable IRF-1hER fusion protein have to be generated. The transfectants should be analysed for proliferation, reversion of the transformed phenotype, expression of MHC class I, MHC class II molecules and cytokine secretion in vivo. To test the direct effect of enhanced IRF-1 activity on tumor growth in vivo, the IRF-1hER transfected cells will be subcutanously injected into SCID or nu/nu mice. Tumor growth will be followed after IRF-1hER activation by estradiol treatment. Chromosomal deletions of the IRF-1 locus in humans are associated with myelodysplasia and certain leukemias. Therefore, it is of interest to investigate the tumor suppressive effects of IRF-1 in hematopoietic tumors. BCR/Abl expressing leukemia cells should be transfected with the IRF-1hER fusion protein and tested for the described functions in vitro and in vivo.
Kröger, A., Dallügge, A., Kirchhoff, S. and Hauser, H.: IRF-1 reverts the transformed phenotype of oncogenically transformed cells in vitro and in vivo, Oncogene, 22, 1045-1056 (2003)
Kröger, A., Ortmann, D., Krohne, T.U., Blum, H., Hauser, H., and Geissler, M.: Growth Suppression of the Hepatocellular Carcinoma Cell Line Hepa1-6 by an Activatable Interferon Regulatory Factor-1 in Mice. Cancer Res., 61, 2609-2617 (2001)
2. Role of NRF and iNOS activity in infection (Hauser)
Nitric oxide (NO) is a mediator of innate immunity that is generated by the inducible isoform of nitric-oxide synthase (iNOS). It exhibits anti-microbial, anti-atherogenic and anti-apoptotic effects. iNOS expression and excessive NO production is observed during many infectious diseases and protection against severe cerebral malaria is associated with increased NO production. iNOS is also strongly upregulated during septic shock. The transcription factor NF- B plays a central role in the induction of iNOS gene transcription. Recently, we have shown that NF- B-repressing factor (NRF), a silencer protein that binds to the negative regulatory element (NRE) in the promoter of the iNOS gene, represses the basal transcription of iNOS. It is still unclear how NF- B and NRF interplay in the regulation of iNOS. In the proposed project the regulation and interaction of both transcription factors upon diverse infection (viral, bacterial) will be investigated and the function and regulation of NRF during infection will be studied. Recently, we have created NRF gene KO mice and cell lines thereof to be used in such studies. The aim is to understand the regulatory mechanisms of the key factor iNOS.
Nourbakhsh, M. and Hauser, H.: Constitutive silencing of IFN-? promoter is mediated by NRF (NF-?B repressing factor), a nuclear inhibitor of NF-?B. EMBO J. 18, 101-111 (1999)
Feng, X., Guo, Z., Nourbakhsh, M., Hauser, H., Ganster, R., Shao, L., and Geller, D.A.: Identification of a negative response element in the human inducible nitric oxide synthase (hiNOS) promoter: The role of NF-kB repressing factor (NRF) in basal repression of the hiNOS gene. Proc. Natl. Acad. Sci., U.S.A., 99, 14212-14217 (2002)
3. Functional characterization of conserved NRF protein domains (Müller/Hauser)
The aim of the project is to understand the regulation of key inflammatory genes at the level of transcription. NF-kB is a transcription factor of central importance in regulating gene activity in inflammation and other processes. Several of the NF-kB regulated genes have been shown to be repressed in the absence of inflammation by a novel factor. We have isolated the gene encoding a NF-kB repressing factor (NRF) from a cDNA library and we presently generate transgenic mice that lack NRF. To investigate the role and potential applications of NRF in NF-kB mediated inflammatory and immune responses, we will establish tissue culture and eventually mouse models. To dissect the function of the protein, the role of individual conserved NRF protein domains will be investigated. Individual domains will be defined in the context of the whole NRF protein or expressed separately or as fusions with other proteins, such as GFP, DNA-binding domains, transcriptional activation domains or histone deacetylases. One goal will be to identify target genes of NRF. In addition, by replacing the NRF DNA-binding domain with the DNA binding domain of other factors that bind to NF-kB regulated promoters, such as IRF-1, it will be tested if specific classes promoters can be modulated and which NRF domains and protein-protein interactions are involved. Dominant mutants will be identified by overexpression of such proteins in wild-type cells. Transcription repressing mutants may allow the suppression of inflammatory reactions in wild-type cells, whereas activating mutants may be used to stimulate the immune system. The use of such mutant proteins in the suppression or induction of immune responses will be investigated. Research focus: Regulation of mammalian genes in inflammatory and antiviral responses; the role intracellular signalling in the control of cell growth and apoptosis; development of tools for efficient recombinant gene expression. Methods: Recombinant DNA technology, PCR, cloning; Recombinant protein expression in E. coli and murine cell culture; Mammalian cell culture, molecular genetics; Protein analysis, ELISA, Western blot, Gel shift; FACS analysis; DNA array chip hybridisation; Visible light and Laser Fluorescence Microscopy.
Feng X, Guo Z, Nourbakhsh M, Hauser H, Ganster R, Shao L, Geller DA. Identification of a negative response element in the human inducible nitric-oxide synthase (hiNOS) promoter: The role of NF-kappa B-repressing factor (NRF) in basal repression of the hiNOS gene. Proc Natl Acad Sci U S A. 2002 Oct 29;99(22):14212-7.
Nourbakhsh M, Kalble S, Dorrie A, Hauser H, Resch K, Kracht M. The NF-kappa b repressing factor is involved in basal repression and interleukin (IL)-1-induced activation of IL-8 transcription by binding to a conserved NF-kappa b-flanking sequence element. J Biol Chem. 2001 Feb 9;276(6):4501-8.
Nourbakhsh M, Hauser H. Constitutive silencing of IFN-beta promoter is mediated by NRF a nuclear inhibitor of NF-kappaB. EMBO J. 1999 Nov 15;18(22):6415-25.
4. In vivo imaging of cell-cell interactions (Köster/Hauser)
Dendritic cells (DCs), the prototypical antigen-presenting cells (APCs), form a link between the first line of host defence and cellular immunity. Present in all tissues, DCs engulf pathogens for antigen presentation and, on migration to draining lymph nodes, they induce differentiation of naïve T cells into effector cells that efficiently combat the pathogen. For example, differentiation of T helper 1 (Th1) cells is triggered by DCs in response to intracellular microbes, whereas Th2-mediated responses eliminate extracellular pathogens. During recognition of specific pathogenic components, such as lipoproteins, lipopolysaccharides or bacterial DNA, an intracellular signalling cascade becomes active leading to the production of regulatory cytokines and the upregulation of MHC and costimulatory molecules. For full activation of T cells a variety of pairs of costimulatory receptors on T cells and their ligands on DCs are required. Generally, costimulatory receptors can be classified into two families: the CD28/ICOS (inducible costimulator) family and the tumor necrosis factor (TNF) receptor family. The use of different intracellular imaging techniques allows to monitor interactions between proteins or changes in protein conformation. Our goal is to analyse cell-cell interaction during the process of pathogenic infection. Optimal T-cell activation by DCs requires intercation via costimulatory molecules. To visualize these receptors on T cells and their ligands on DCs we will fuse them to different variants of GFP. Expression of the tagged molecules on DCs and T cells allows to analyse interaction between these receptors in individual living cells. In a first set of experiments this will be done by Fluorescence recovery after Photobleaching (FRAP) for different costimulators molecules. FRAP allows to determine the mobility of proteins in the extracellular membrane. If these receptors interact with their counterparts on target cells their mobility within the membrane should be reduced. Different receptor-ligand pairs can be analysed answering questions about the kinetics of interaction. In a second part interacting adaptor molecules can be analysed by FRAP and Fluorescence Resonance Energy Transfer to follow the initiation of signalling.