Abstract
Abstract: :
Purpose: To determine if Akt plays a role in the phosphorylation and activation of telomerase in canine lens epithelial cells (LEC), and to determine whether inhibition of telomerase activity, via Akt inhibition, causes LEC to be more susceptible to ultraviolet–B (UV) irradiation. Methods: Primary LEC cultures were grown to 90% confluence. Cells were treated with two Akt inhibitors at 0, 5 or 10 µM for 24 hours; control plates only received inhibitor vehicle. In addition, cultured LEC were exposed to acute UV at either 0 or 150 J/m2. Whole lenses were treated with 10µM Akt inhibitors for 7 days and exposed to chronic UV at either 0 or 150 J/m2 every other day for 3 exposures. Lenses were also examined daily for cataractous changes. Cells or anterior lens capsules with adherent LEC were harvested, protein extracted and quantified by Bradford assay. Telomeric repeat amplification protocol (TRAP)–ELISA assay was performed to determine telomerase activity. Telomerase activity was measured at 450 nm with reference of 690 nm. Reaction products were also run on 12% PAGE gels with TRAP controls as a semi–quantitative method of telomerase activity to confirm ELISA data. Results: 10uM of both Akt inhibitors significantly decreased telomerase activity (p<0.05). LEC treated with 10µM Akt inhibitor and exposed to UV irradiation were more susceptible to UV induced DNA damage and apoptosis. Whole lenses treated with 10 µM Akt inhibitor and exposed to UV developed cataractous changes; untreated unexposed control lenses and lenses exposed to UV alone without Akt inhibitor did not develop cataractous changes after 3 exposures. Conclusion: Cells with telomerase activity are either regulated by protein kinase C (PKC) or Akt. Previous experiments in canine LEC found PKC inhibition ineffective in lowering telomerase activity, therefore, we were interested in determining whether Akt regulated telomerase in LEC. We found that 10µM Akt inhibitor decreased telomerase activity. When cells or lenses were exposed to UV, they were unable to repair or recover from the damage and whole lenses developed cataractous changes. Based on the data, Akt is necessary for telomerase activation and telomerase protects LEC against UV induced oxidative damage.
Keywords: protein structure/function • radiation damage: light/UV