Although reactive oxygen intermediates (ROI) has long been suspected to play a central role in cell killing by radiation and chemotherapeutic agents, specific target genes responsible for ROI mediated cell death or survival have not been identified. Elucidation of ROI mediated signaling may provide new gene targets for enhancing tumor cell sensitivity to anticancer therapy. Recent results suggest that the mitochondrial antioxidant enzyme, manganese-containing superoxide dismutase (MnSOD), is induced by exposure of transformed human cells to ionizing radiation. Additional evidence demonstrates that expression of the gene that encodes MnSOD may be via the stress responsive transcription factor NF-kappaB. Analysis of gene expression profiles in irradiated cells and cells over expressing MnSOD has identified two key pro-survival genes, cyclin B1 and 14-3-3zeta, that can be inhibited by blocking MnSOD expression and were not induced by radiation in Sod2 knockout (-/-) cells. Moreover, inhibiting NF-kappaB using mutant IkappaB, inhibited expression of MnSOD as well as cyclin B 1 and 14-3-3zeta. Therefore, a signaling network involved NF-kappaB, MnSOD, and a group of pro-survival effector genes appears to be activated and required in signaling cell sensitivity to ROI mediated cytotoxicity. This proposal will test the hypothesis that changes in steady state levels of H2O2 caused by MnSOD plays a key role in regulating pro-survival genes cyclin B1 and 14-3-3zeta and if blocking cyclin B1 and 14-3-3zeta gene expression increases cell sensitivity to radiation-induced cytotoxicity.
Four Specific Aims will test this hypothesis: a) Determine if reconstitution of MnSOD function in mitochondria of Sod2-/- and MCF-7 cells restores cyclin B1 and 14-3-3zeta expression and if redox-sensitive transcription factors are responsible for MnSOD mediated activation of gene promoter for cyclin B 1 and 14-3-3zeta transcription; b) Determine if MnSOD-induced changes in steady state levels of mitochondrial H2O2 contribute to alterations in the expression of cyclin B1 and 14-3-3zeta using replication incompetent adenoviral vectors targeting the H2O2 scavenging enzymes, catalase (CAT) or glutathione peroxidase (GPx), to mitochondria; c) Determine if redox sensitive subunits of transcription factors (AP-1, STAT and p300) control MnSOD-induced cyclin B1 and 14-3-3zeta expression by analysis of the rate of cJun and STAT phosphorylation and p300 interaction with cJun and STAT; and d) Determine if cyclin B1 and/or 14-3-3zeta overexpression is one of the key signaling pathways required for the radioresistance phenotype. Stable MCF-7 and HK18 cells lines overexpressing cyclin B 1 and 14-3-3zeta will be selected and resistance to radiation-induced toxicity tested. Then resistant clones from radiation treated MCF-7 and HK18 cells as well as MCF+SOD and HK18+SOD cells will be exposed to siRNA to cyclin B1 or 14-3-3zeta to determine if the radioresistant phenotype can be reversed.
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