The overall goal of this research is to define the molecular mechanisms for the regulation of MnSOD expression in normal and tumor cells. It has been shown that most tumor cells have reduced MnSOD activity when compared to their normal counterparts and that the expression of MnSOD suppresses neoplastic transformation. Previous studies have shown that the reduced MnSOD activity in tumor cells is largely due to defects in the transcription of the MnSOD gene. Genomic DNA cloning and sequencing has identified several potential cis-acting regulatory DNA elements in the MnSOD gene isolated from normal human cells. In this application, we will investigate whether the reduction of MnSOD transcription in tumor cells is due to defects in the cis-acting regulatory DNA elements of MnSOD gene or to defects in the transactivating factors which control the expression of the MnSOD gene in tumor cells. This investigation will be undertaken in three stages. First, the genomic DNA including the upstream and downstream sequences in human cancer cells will be compared to that in normal human cells by DNA amplification, cloning, and sequencing methods, Second, the cis-acting elements in the human MnSOD gene which control the transcription of human MnSOD under normal growth conditions and under oxidative stress induced by cancer therapy and cancer promoting agents will be identified using systematic deletion and mutation analyses of the human MnSOD gene. Third, the presence of transcriptional activator/suppressor proteins which affect the constitutive and inducible expression of human MnSOD in normal and tumor cells will be determined by transfection and DNA binding analyses. The results obtained from this study should direct the development of strategies to modulate MnSOD activity in normal and tumor cells for the control of cancer development and the treatment of developed malignancies.
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