MECHANISM OF NF-KB ACTIVATION BY DNA DAMAGE

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DESCRIPTION (As Adapted From the Investigator's Abstract): DNA damaging agents are an important component of anti-cancer therapy. These agents elicit numerous cellular responses, including the induction of apoptosis. Recent data suggest that the NF-kappaB transcription factor activation may have anti-apoptotic effects. Understanding the mechanisms of NF-kappaB activation may lead to improved tumor killing by attenuating downstream antiapoptotic. However, the exact mechanism of NF-kappaB activation after specific DNA damage is unknown. This proposal focuses on the mechanisms of NF-kappaB activation by camptothecin (CPT-related anticancer agents, which are Topoisomerase (Topo) I inhibitors. Inactive NF-kappaB is present in the cytoplasm and its activation requires nuclear translocation. How is cytoplasmic NF-kappaB activated by nuclear DNA damage? Studies in this laboratory strongly suggest the presence of a nuclear to cytoplasmic signaling pathway. Three Specific Aims are proposed to test the hypothesis that Topo-I generated DNA double-stranded breaks in the nucleus initiate a signaling pathway that ultimately activates IkappaB kinase to release NF-kappaB from its inhibitor IkappaB. Two aims will specify the nuclear signaling events triggered by CPT treatment by focusing on the role of DSB (Aim I) and DNA-dependent protein kinase (DNA-PK, Aim 2). Cell cycle expression of an NF-kappaB-responsive reporter and restriction enzyme mediated generation of DSB will determine whether DSB is sufficient for NF-kappaB activation. The critical involvement of DNA-PK will be uncovered by assaying the NF-kappaB activity in genetically matched cells from mice deficient for each of the kinase complex components. Finally the endpoint of the signaling in the cytoplasm will be specified by focusing on the CPT-dependent regulation of an IkappaB kinase complex that phosphorylates the inhibitor IkappaB to ultimately release active NF-kappaB (Aim 3). Aim I will determine the target specificity of Topo-I-mediated DNA damage to trigger NF-kappaB activation.