| The NF-kappaB/Rel family of transcription factors regulates genes important in inflammation, the immune response, cell growth and survival. Moreover, misregulation of the NF-kappaB signaling pathway occurs in many human diseases including immunodeficiencies, chronic inflammation, heart disease, and cancer. For example, mutations in the scaffolding protein NEMO have been identified in patients suffering from various immunodeficiencies. NEMO is an adaptor protein that controls the activity of the IkappaB kinase complex. In this thesis, it is shown that NEMO can exist as a disulfide-bonded dimer in cells and that formation of this dimer can be modulated by hydrogen peroxide. Site-directed mutagenesis of NEMO has identified residues Cys54 and Cys347 as being required for disulfide bond formation. It is demonstrated that the Cys residues required for disulfide-mediated dimer formation of NEMO are also required for full activation of NF-kappaB in response to tumor necrosis factor. Furthermore, a computer-derived structural model of the NEMO dimer that accommodates these disulfide bonds is proposed. Genetic alterations in the gene encoding the transcription factor REL have been identified in certain human B-cell lymphomas, including many diffuse large B-cell lymphomas (DLBCLs), the most common form of non-Hodgkin's lymphoma. DLBCL can be divided into two molecular and clinical subgroups: a germinal center B-cell (GCB) subtype and an activated B-cell (ABC) subtype. In this thesis, gene expression profiling is used to show that overexpression of an oncogenically-activated form of REL, RELDeltaTAD1, in a GCB-like DLBCL cell line (BJAB) can convert these cells to an ABC-like DLBCL cell line. BJAB-RELDeltaTAD1 cells also show increased tumor forming ability in immunodeficient mice and are more resistant to doxorubicin-induced apoptosis. Finally, a molecular mechanism for the transcriptional control of the gene encoding the GCB DLBCL marker CD10 is delineated. It is demonstrated that the transcription factor PU.1 can directly bind to a site in the CD10 promoter/enhancer to increase transcription. Moreover, it is shown that upregulation of NF-kappaB induces expression of the microRNA miR-155, which is a known regulator of PU.1. Taken together, these results indicate that activation of NF-kappaB results in increased levels of miR-155 and consequently reduced levels of PU.1 and CD10. |
