Mutations in the promoter reveal a cause for the reduced expression of thehuman manganese superoxide dismutase gene in cancer cells

Manganese superoxide dismutase (MnSOD) has been shown to play an important role in preventing the development of cancer. MnSOD activity is reduced in many transformed cells and tumor tissues. We previously showed that the red uced level of MnSOD activity in cancer cells was not due to a defect in the primary structure of MnSOD protein, but rather was due to defects in gene expression. To elucidate the cause for the reduced expression of human MnSO D in cancer, we investigated the nucleotide sequence in the regulatory regi on of the MnSOD gene in a normal human cell line and various human tumor ce ll lines. A DNA fragment spanning 3.4 kb 5' flanking region of the MnSOD ge ne isolated from a normal human genomic DNA library was used to determine t he DNA sequence of MnSOD promoter. PCR primers were used for amplification of the 3.4 kb 5' flanking region of the human MnSOD gene in cancer cells. S equence analysis identified three heterozygous mutations in the proximal re gion of the promoter in five human tumor cell lines; These mutations, clust ered around the GC-rich region of the human MnSOD promoter, change the bind ing pattern of AP-2 and lead to a reduction in transcription activity using a luciferase reporter assay system. These results suggest that the reduced level of MnSOD expression in some tumor cells is, at least in part, due to a defect in the DNA sequence of the promoter region.