Enzymatic activity is necessary for the tumor-suppressive effects of MnSOD

The antioxidant protein manganese-containing superoxide dismutase (MnSOD) has been found to be a new type of tumor-suppressor protein. Overexpression of the cDNA for this gene in various types of cancer via plasmid transfection or adenovirus transduction leads to growth suppression both in vitro and in vivo. The growth-suppressive effect of MnSOD overexpression has been presumed to be due to the enzymatic activity of the MnSOD protein, but could be due to a number of other mechanisms, including a regulatory effect of the RNA or protein produced. To examine this question, we used site-directed mutagenesis to produce a mutant form of human MnSOD that has a leucine at amino acid 26 in the active site rather than the usual histidine. We demonstrate that plasmid transfection or adenoviral transduction of this mutant MnSOD cDNA leads to a large increase in immunoreactive MnSOD protein, but little or no increase in enzymatic activity. In contrast, overexpression of wild-type MnSOD leads to cells with both increased MnSOD protein and activity. Overexpression of wild-type, but not mutant, MnSOD leads to decreased plating efficiency and growth. These results clearly demonstrate that the tumor-suppressive effect of MnSOD protein is largely due to its enzymatic activity.