Abstract
AbstractLead compounds are ubiquitous environmental contaminants that induce cellular transformations in cultured human cells and cancers in experimental rodents. Epidemiological studies suggest that lead exposure increases risk of lung, stomach, and bladder cancers in smelter or battery workers. Lead exhibits weak mutagenicity in mammalian cells and elicits signal transduction pathways in many aspects. To gain insight into the mechanism that lead-induced signal molecules affect genotoxicity, in this thesis, we investigated whether lead acetate (Pb(II)) could elicit four MAPKs and AKT to regulate DNA repair, cytotoxicity and mutagenicity in mammalian cells. We found that Pb(II) increases the phosphorylated-ERK1/2 and phosphorylated-AKT but not the phosphorylated-ERK5, phosphorylated-p38 and JNK activity in human non-small cell lung adenocarcinoma CL3 cells. The duration of ERK1/2 activation is much longer than AKT activation and these two signals are independently activated by Pb(II) in CL3 cells. Intriguingly, a MKK1/2 inhibitor PD98059 markedly blocks ERK1/2 activation and greatly promotes the hprt mutation frequency and cytotoxicity in CL3 cells as well as diploid human fibroblasts treated with Pb(II). Conversely, inhibition of the AKT signal by wortmannin does not exhibit such effects. Mannitol, a hydroxyl radical scavenger, blocks the ERK1/2 activation by Pb(II) and prevents the genotoxicity induced by Pb(II) in the presence of PD98059. The extent of Pb(II) genotoxicity is negative proportional to the cellular amounts of apurinic/apyridimic endonuclease (APE). In addition, Pb(II) increases the endonuclease activity of APE, which can be reduced by PD98059 co-treatment, suggesting that Pb(II)-activated ERK1/2 enhances cellular base excision repair (BER). The Pb(II)-induced genotoxicity is also significantly higher in nucleotide excision repair (NER)-deficient cells than their counterpart cells. Furthermore, cellular NER synthesis is enhanced by Pb(II) exposure, which is markedly suppressed by PD98059. Activation of ERK1/2 by expressing a constitutively active form of MKK1 in CL3 cells also elevates cellular NER synthesis. By using a cDNA microarray technique, we found that Pb(II) increases the expression of genes functioning in DNA repair, antioxidant, and metastasis in ERK1/2-dependent manner. Together, these results indicate that Pb(II) elevates cellular ROS resulting in genetic damage as well as activation of the sustained-ERK1/2, thereby triggering BER, NER and antioxidant systems to prevent cytotoxicity and mutagenicity, however, Pb(II)-elicited ERK1/2 may also promote metastasis.We further investigated the mechanism by which sustained ERK1/2 activation is established by Pb(II). We demonstrated that the Ras-Raf-MKK1/2 signaling cannot fully account for the sustained ERK1/2 activation elicited by Pb(II). It is intriguing that Pb(II) treatment reduces MKP-1 protein levels in time- and dose-dependent manners, which correlates with sustained ERK1/2 activation, and that Pb(II) also induces mRNA and de novo protein synthesis of MKP-1. In Pb(II)-treated cells, MKP-1 is poly-ubiquitinated, and proteasome inhibitors markedly alleviate the ubiquitination and degradation of MKP-1. PD98059 greatly suppresses MKP-1 ubiquitination and degradation. Conversely, constitutive activation of MKK1/2 triggers endogenous MKP-1 ubiquitination and degradation. Furthermore, expression of functional MKP-1 decreases ERK1/2 activation and enhances cytotoxicity under Pb(II) exposure. Analysis of the MKP-1 amino acid sequence reveals that a D box to be recognized by ubiquitin-proteasome system located in position 278-286, and two putative ERK1/2 phosphorylation sites (Ser296 and Ser323) located upstream of the ERK1/2 docking, DEF motif (FXFP, 339-342). By using site-direct mutagenesis technique, we found that the D box and Ser296 may be correlated with MKP-1 degradation. Taken together, these results indicate that activated ERK1/2 can trigger MKP-1 phosphorylation and degradation via the ubiquitin-proteasome pathway, thus facilitating long-term activation of ERK1/2 against cytotoxicity.