PURPOSE

To determine the effect of changes in ferritin subunit makeup on Fe metabolism and the resistance of lens epithelial cells (LEC) to photo-oxidative stress.

METHODS

Cultured canine LEC were transiently transfected with pTargeT mammalian expression vector containing the whole coding sequence of H- or L-chain cDNA. The subunit composition of newly synthesized ferritin was analyzed by metabolic labeling and SDS-PAGE electrophoresis. Total ferritin concentration was measured by ELISA: Fe uptake and incorporation into ferritin was determined by incubating transfected cells with (59)Fe-labeled transferrin followed by native PAGE electrophoresis. The effect of UV irradiation was assessed by cell count after exposure of transfected cells to UVB radiation.

RESULTS

Transfected cells differentially expressed H- and L-ferritin chains from cDNA under the control of CMV promoter; overexpression of L-chain was much greater than that of H-chain. The expressed chains assembled into ferritin molecules under in vitro and in vivo condition. The ferritin of H-transfectants incorporated significantly more Fe than those of L-transfectants. The UVB irradiation reduced cell number of L-transfectants by half, whereas H-chain transfectants were protected.

CONCLUSIONS

Post-transfectional expression of ferritin H- and L-chains in LEC appears to be regulated differentially. Overexpression of L-chain ferritin did not have a major effect on cellular Fe distribution and did not protect LEC against UV irradiation, whereas overexpression of H-chain resulted in increased storage of Fe in ferritin and protected cells from UV damage.