Abstract
In this thesis, we have firstly investigated the roles of ERK and p38 MAPK signal transduction pathways induced by cadmium (Cd) in genotoxicity, apoptosis and cell cycle progression. Exposing asynchronous CL3 cells to CdCl2 for 2 h (45% viability) caused irreversible mitotic arrest. Exposing synchronous early-G2 cells to Cd markedly delayed mitotic exit and subsequently induced sub-G1 populations; however, this did not alter the levels of Cdc2 and cyclin B1. These results suggest that Cd elicits mitotic arrest without affecting the progression of G2 to mitosis. Using counterflow centrifugal elutriation and flow cytometry analysis, CL-3 cells synchronized at G1-, S-, and G2/M-phases were collected and treated with CdCl2. G2/M was the most sensitive cell cycle phase to Cd for the induction of ERK and p38 MAPK activities, cytotoxicity, apoptosis, micronucleus, and intracellular peroxide; despite that similar Cd accumulation was observed in G1-, S-, and G2/M-cells. Co-treatment early-G2 cells with Cd and SB202190, an inhibitor of p38 MAPK, significantly decreased the induction of micronucleus, mitotic arrest, and apoptosis. Conversely, PD98059, an inhibitor of the ERK upstream activators MKK1/2, enhanced micronucleus and apoptosis in Cd-treated early-G2 cells. Together, the results suggest that intracellular peroxide may participate in the activation of ERK and p38 MAPK by Cd; also, the activated-p38 MAPK may contribute to mitotic arrest and genome instability, whereas the activated-ERK may help to maintain genome integrity and survival. Upon cells exposure to stress, the tumor suppressor p53 is rapidly activated to surveillance DNA damage, trigger cell cycle arrest or apoptosis. In this thesis, we have further explored the ability of Cd to activate p53 and the mechanisms involved. We have also investigated the interaction between p53 and MAPK signals as well as their roles in apoptosis and cell cycle arrest induced by Cd. Exposure CL3 cells to Cd for 2 h markedly induced the levels of p53, phospho-p53 (Ser-15) and p53 downstream gene products p21, BAX, MDM2 and GADD45 and the induced levels further increased at 2-8 h after removing Cd from media. Cd did not induce phospho-p53 (Ser-15) and only slightly increased p53 protein in GM03395 fibroblasts derived from a patient with mutation in ataxia telangiectasia (ATM), suggesting the induction of phospho-p53 (Ser-15) by Cd requires ATM. Conversely, Cd could activate p38 and ERK in ATM-/- or H1299 (p53-null) cells, suggesting the ATM-p53 pathway is not necessary for the activation of MAPKs in Cd-treated cells. L-NAME, a NO synthase inhibitor, dose-dependently decreased the Cd-elicited p53, phospho-p53 (Ser-15) and phospho-p38 in CL3 cells as well as HFW diploid human fibroblasts, suggesting NO synthase or NO are involved in Cd-elicited signals. Cd could activate caspase-3 and apoptosis in both CL3 and H1299 cells. Neither SB202190 nor PD98059 affected Cd-induced phospho-p53 (Ser-15), p53 and BAX protein levels in CL3 cells. SB202190 decreases the sub-G1 fraction and the activation of caspase-3 induced by Cd in both CL3 and H1299 cells. However, SB202190 suppresses and enhances p21 expression in Cd-treated CL3 (p53-proficient) and H1299 (p53-null) cells, respectively. Cd caused G2/M arrest in CL3 but not H1299 cells, although H1299 cells were slightly sensitive than CL3 cells to Cd-induced cytotoxicity. SB202190 enhanced M exit in Cd-treated CL3 cells, while it enabled G2/M arrest in Cd-treated H1299 cells. These results suggest Cd-elicited p38 MAPK up-regulates p21 in p53-proficient cells to facilitate G2/M arrest, while p38 suppresses p21 in Cd-treated p53-null cells and thereby bypass G2/M arrest. Together, p38-triggered apoptosis is independent of the p53-BAX pathway, while p38-mediated G2/M arrest or bypass through up- or down-regulation of p21 is p53 dependent upon cells exposure to Cd. In the last portion of this thesis, we have adopted a stable human cell line H1299-tsp53, which expressing a temperature-sensitive p53 protein p53Leu173 to further explore the role of p53 in Cd-induced cell cycle arrest and apoptosis, and its co-regulation of downstream effectors with p38 MAPK activity. Switching cell cultures from the restrictive temperature 37℃ to the permissive temperature 32℃ can tightly and reversibly regulate the transcriptional activity of p53 from non-functional to functional status. H1299-tsp53 cells cultured at 32℃ for 18 h were markedly more resistant to the induction of cytotoxicity, micronucleus, sub-G1 fractions and caspase-3 activation by Cd (40-120 mM) than those cultured at 37℃, suggesting a protective role of p53 in cells exposed to Cd. H1299-tsp53 cells expressing functional p53Leu173 protein had significantly higher levers of p21 and MDM2 proteins than those expressing mutant form of the p53Leu173 protein. Expression of functional p53Leu173 protein exhibited slower proliferation rate and a longer length of cell cycle, which may due to the high levels of p21. However, Cd could also induce p21 in cells cultured at the restrictive temperature. Furthermore, mutant p53Leu173 protein is much sensitive than the functional form to Cd in the induction of phospho-p53 (Ser-15), phospho-p38 and phospho-ERK and degradation of pro-caspase-3. SB202190 protected the degradation of pro-caspase-3 in Cd-treated cells, whereas, PD98059 slightly enhanced it. The effects of these kinase inhibitors were stronger in Cd-treated cells had mutant p53Leu173 protein. Cd also markedly induced the survival phospho-AKT signal in a p38-dependent and p53-independent manner, which is possibly due to negative feedback control. SB202190 completely suppress the levels of phospho-AKT induced by Cd, whereas PD98059 did not affect it. Together, results shown here suggest that p53 is required for cell cycle arrest, while p38 triggers apoptosis in cells after exposure to Cd. In summary, we have characterized that the activation of ERK, p38 MAPK, p53 and p21 play essential roles in regulating signals involved in mitotic arrest, apoptosis and genotoxicity induced by Cd.