Effects of overexpression of GMF on cell growth and redox regulation

GMF (glia maturation factor) is a 17-kDa protein endogenous to the brain. The protein is normally not secreted by the cells and appears to have important intracellular functions. The protein is rapidly and transiently phosphorylated when a cell is simulated by a phorbol ester. GMF contains many phosphorylation sites and can be phosphorylated by at least four kinases: by PKA at T26 and S82, by PKC at S71, by p90 RSK at T26, and by CKII at S52. In an in vitro assay, PKA-phosphorylated GMF shows a strong inhibition on ERK 1/2 MAP kinase and a strong enhancement on p38 MAP kinase. In order to evaluate the intracellular functions of GMF, we overexpressed GMF in C6 glioma cells using two methods: stable transfection using pcDNA3 plasmid, and transient transfection using replication-defective human adenovirus. With both methods, C6 cells transfected with GMF and overexpressing the protein exhibit a lower saturation density in culture compared to non-transfected or vector alone controls. Transfected cells also exhibit morphological differentiation such as the outgrowth of cell processes. When inoculated into nude mice, transfected cells are less tumorigenic than controls. In tissue culture, transfected cells show a 3.5-fold increase in CuZn-dependent Superoxide dismutase (CuZnSOD) activity, but not in MnSOD activity. Western blot analysis reveals a 3.5-fold increase in CuZnSOD protein, suggesting an enzyme induction.