Abstract
Abstract Apurinic/apyrimidinic endonuclease 1 (APE1)/redox effector factor-1 is a multifunctional enzyme involved in DNA base excision repair and protein redox regulation. Previous reports of our laboratory have showed that lead acetate (Pb) elicits EGFR activation to initiate the SFK/PKC/Ras/Raf-1/MKK1/2/ERK signaling cascade functioning against genotoxicity. In this thesis, I explored whether APE1 is involved in the regulation of oxidative and nitrosative stresses that may affect ERK signaling and cell cycle progression following Pb exposure. I found that Pb induced APE1, iNOS expression and ROS/NO generation and caused ERK oxidation and S-nitrosylation in CL3 human lung cancer cells. The Pb-elicited ROS levels and cytotoxicity were further enhanced by introducing small interfering RNA specific for APE1 (siAPE1). E3330, an inhibitor of APE1 redox activity, also augmented the ROS/NO levels and cytotoxicity in Pb-treated cells. Intriguingly, transfected siAPE1 or E3330 co-treatments increased ERK oxidation and S-nitrosylation but abolished the kinase activity; conversely, forced expression of APE1 up-regulated the ERK activation by Pb or serum in both Cys65-redox activity dependent and independent manners. Moreover, APE1 formed complex with ERK2, and its redox activity prevented ERK oxidative or nitrosative inactivation. APE1 redox activity also facilitated the Cyclin D1 expression and G1-to-S progression following Pb exposure. In summary, the results suggest that the APE1 redox regulation domain has a de-oxidation and de-nitrosylation activity for ERK2 and thereby maintaining the kniase activity to promote cell proliferation.