Abstract
Lead compounds are widely distributed environmental toxicants that can cause genotoxicity and cytotoxicity, and are probably carcinogenic to humans. Imbalanced intracellular nitric oxide and reactive oxygen species have been associated with cancer development. Previous studies have indicated that ERK1/2 activation elicited by lead acetate (Pb) enhances nucleotide excision repair synthesis and thereby decreases cytotoxicity and mutagenicity. APE1, also known as Ref-1, has the main functions in base excision repair and redox activation of transcription factors. However, some cancer cells have a higher protein level of APE1. This thesis focuses on investigating the roles of nitric oxide and APE/Ref-1 in ERK1/2 activation elicited by Pb in H157 lung squamous-cell carcinoma cells. The analysis using flow cytometry in combined with fluorescent dye showed that Pb could enhance the generation of nitric oxide and reactive oxygen species in H157 cells. Immunoblotting showed that Pb induced ERK1/2 phosphorylation, which indicates the activation of this signaling. Pb could also promote S-nitrosylation of ERK as determined by using SNO-RAC in combined with immunoblotting. The nitric oxide donor S-nitrosoglutathione induced ERK1/2 phosphorylation and its co-treatment with Pb additively increased the signaling level. Conversely, the nitric oxide synthase inhibitor L-NAME could reduce the Pb-induced ERK1/2 phosphorylation. Additionally, Pb increased APE1 protein levels in H157 cells. Depletion of APE1 by using small interfering RNA (si-APE1) resulted in a decrease of ERK1/2 phosphorylation in Pb-treated H157 cells. Conversely, forced co-expression of APE1 and constitutive activation MKK1─ERK1/2 signaling in H157 cells showed that APE1 enhanced ERK1/2 activation. In this thesis, I also established several stable cell lines that expressing regulatable APE1-specific interfering RNA. Together, the results indicate that the nitric oxide and APE1 levels are positively correlated with ERK1/2 phosphorylation in H157 cells following Pb, and the exposure also facilitates ERK1/2 S-nitrosylation. The results obtained from this thesis should provide basis for the future study of the impact of S-nitrosylation on ERK1/2 signaling.