) The long-term goal of this application is to characterize the mechanisms by which mitochondrial superoxide regulates the malignant phenotype of fibrosarcoma cells in vitro and in vivo. Preliminary studies conducted by the applicant demonstrate that manganese superoxide dismutase (MnSOD) overexpression, an enzyme which catalyzes the removal of superoxide from the mitochondria, is inversely correlated with cellular growth inhibition. In addition, the levels of IL-1-alpha specifically respond to changes in MnSOD activity. When MnSOD levels are increased two- to four-fold or cells are cultured in low oxygen (3 percent), a decrease in both basal and tumor necrosis factor (TNF)-induced IL-l-alpha is observed. However, as MnSOD activity increases further, a corresponding increase in IL-l-alpha is observed with an associated decrease in the growth of the fibrosarcoma cells. Both the growth inhibition and the increase in IL-l-alpha levels in cells which highly overexpress MnSOD (10-15-fold) can be blocked by growth in three percent oxygen. This latter finding implies that the effects of MnSOD overexpression on cell growth and IL-l-alpha levels are mediated by mitochondrial superoxide. Nuclear run-on, promoter, and NFkB-DNA binding activity analysis demonstrate that the alterations in the steady state levels of IL-1-alpha mRNA by either MnSOD overexpression or growth in low oxygen are not accompanied by changes in IL-1-alpha transcription. The candidate has also demonstrated that MnSOD overexpression or oxygen alter IL-l mRNA levels by modulating its stability. Under low oxygen or a subtle increase in the MnSOD activity of the fibrosarcoma cells, IL-1 mRNA turnover is increased. Conversely, under 21 percent oxygen or high MnSOD overexpression IL-l-alpha mRNA becomes stable. He proposes to characterize the human IL-l-alpha mRNA and hence define the putative superoxide responsive region in the mRNA. A detailed deletion and mutation analysis of the mRNA will be conducted utilizing both the 5' and 3'-untranslated regions of the IL-l-alpha mRNA ligated into a reporter gene construct and transiently transfected into HT-1080 fibrosarcoma cells. These studies will establish the specific regions of the IL-l-alpha mRNA that are necessary for regulation by oxidants. He will further address the involvement of superoxide sensitive RNA-binding proteins once he has defined the regions of the RNA which respond superoxide. He also proposes to characterize the changes in the mitochondrial oxidant production and integrity which lead to alterations in IL-1-alpha levels and cell growth in response to MnSOD overexpression. IL-1-alpha is a major inflammatory cytokine which has been shown to mediate tumor regression by enhancing host antitumor defense mechanisms. Thus, he will investigate if tumor regression associated with MnSOD overexpression in vivo is mediated in part by IL-1-alpha. These studies should not only shed light on the mechanisms which mediate the reversal of tumor growth by MnSOD but also define this newly discovered mode of regulation of IL-1-alpha.
|Milne, Roger L; Lorenzo-Bermejo, Justo; Burwinkel, Barbara et al. (2011) 7q21-rs6964587 and breast cancer risk: an extended case-control study by the Breast Cancer Association Consortium. J Med Genet 48:698-702|