Superoxide dismutase activities of differentiating clones from an immortal cell line

We have studied superoxide dismutase (SOD) levels in the X-REF-23 rat embryo fibroblast cell line. X-REF-23 is an immortal cell line that maintains a nontransformed phenotype throughout its known lifespan. Low-passage X-REF-23 cells undergo spontaneous differentiation into muscle and adipose cells, while high-passage X-REF-23 cells undergo little or no differentiation. SOD activities were measured in subclones of X-REF-23, which differentiate into muscle (AMC subclone) or adipose (AMB-J) cells, as well as the parental nondifferentiating X-REF-23 cells. Total SOD activity increased in all three cell lines with time in culture. Cu-ZnSOD was induced in the AMB-J and the X-REF-23 cells with time in culture, whereas the AMC cells showed no induction. MnSOD activity was induced during the time period in which differentiation occurred in the two differentiating clones. In contrast, MnSOD was not induced in this time period in the nondifferentiating X-REF-23 cell line. However, MnSOD activity was induced in the latter cell line at a much later time. Levels of immunoreactive MnSOD correlated quite well with MnSOD activity in all three cell lines. The nondifferentiating X-REF-23 cells, but not the two differentiating cells lines, showed a large increase in cell organelles with time in culture. In particular, an increase in very small mitochondria was observed; these mitochondria often showed evidence of disorganization.