| The ala16val polymorphism, found in the mitochondrial targeting signal of manganese superoxide dismutase (MnSOD), has been associated with a number of diseases, including breast cancer. MnSOD is an important antioxidant enzyme that functions in the mitochondrial matrix to dismutate superoxide to hydrogen peroxide. A single nucleotide polymorphism causes a change in the amino acid sequence from alanine (ala) to valine (val) at the 16th amino acid and is referred to as the alal6val polymorphism. Previous studies have reported that the polymorphism may cause an increase in import of MnSOD to the mitochondrial matrix in vitro. Altered levels of MnSOD due to the polymorphism are hypothesized to disrupt the oxidative balance in the cell, making the cell more susceptible to oxidative stress. We hypothesized that the alal6val polymorphism alters MnSOD protein levels and investigated this hypothesis in human breast epithelial cell lines and human cryopreserved hepatocytes. In the hepatocytes, we found no association between MnSOD protein, activity, or mRNA levels and the alal6val polymorphism. The breast epithelial cell lines showed that some, but not all, of the ala/ala lines had increased levels of MnSOD protein, activity, or protein per unit mRNA compared to the val/val cell lines. In order to assess the role of this polymorphism in situ, human MnSOD was cloned into the pcDNA3.1/Zeo expression vector and stably transfected into mouse embryonic fibroblasts (MEF) developed from the MnSOD knockout mouse. Six cell lines were established, 3 that express MnSOD-ala protein, and 3 that express MnSOD-val. MnSOD enzymatic activity correlated fairly well with MnSOD protein levels determined by Western blot. MnSOD mRNA expression, evaluated by TaqMan(TM) real-time PCR, was significantly lower in MnSOD-ala lines versus MnSOD-val lines (p<0.05). The MnSOD-ala lines produced significantly more MnSOD protein per unit mRNA than the MnSOD-val lines (p<0.05). This suggests that the MnSOD-ala lines are more efficient in their production of MnSOD protein than are MnSOD-val lines. MnSOD-val lines had significantly higher copy numbers of MnSOD DNA than did the MnSOD-ala lines. There was no association between the amount of MnSOD activity and production of superoxide or hydrogen peroxide, as detected by flow cytometry. |
