Date of Award
2005
Degree Type
Dissertation
First Advisor
Zahir A. Shaikh
Abstract
Cadmium (Cd) may induce renal toxicity by modifying the activity of oxidative stress sensitive transcription factors that are responsible for regulating apoptotic gene expression and the genes that modulate the cell cycle. The present study was designed to elucidate the involvement of two such transcription factors, NF-κB and p53, in Cd-induced apoptosis and cell cycle arrest. Cells from a normal rat kidney epithelial cell line, NRK-52E, were incubated with CdCl2 for 5h and maintained in Cd-free medium for an additional 12h. The DNA binding activity of NF-κB was decreased by Cd in a concentration- and time-dependent manner. Pretreatment of the cells with antioxidants, U8386E or BHT, preserved NF-κB activity. Cd treatment also decreased the activity of TNF-α-induced IκB kinase α. Phosphorylation of IκBα and NF-κB p65 were reduced by Cd as well. Furthermore, Cd exposure resulted in a decline in protein level of the NF-κB target gene products, cIAP-1 and cIAP-2. Consequently, caspase-3, -7 and -9 were activated and the cells exhibited apoptotic DNA fragmentation. Overexpression of NF-κB-p65 by transfection of NF-κB-p65 EGFP plasmid protected the cells from Cd-induced apoptosis. Moreover, attenuation of NF-κB activity by pretreatment with SN50, a NF-κB translocation inhibitor, potentiated the apoptosis. These results suggested that in the NRK52E cells NF-κB plays an important role in Cd-induced apoptosis. NRK-52E cells were treated for up to 24h with CdCl2 in DMEM containing 10% calf serum. Flowcytometric analysis revealed a significant time- and concentration-dependent increase in cells in G2/M cell cycle phase. Treatment with 20 μM Cd for 24h doubled the number of cells in this phase. The cell cycle arrest was related to the decrease in cyclins A and B. Additionally, Cd treatment led to down-modulation of cyclin-dependent kinases, cdk2 and Cdc2. Cd also elevated the expression of cyclin-specific inhibitor KIP1/p27 and WAF1/p21. Moreover, wild type p53 DNA binding activity was up-regulated upon Cd exposure. These observations suggest that Cd-induced cell cycle dysregulation is mediated via modulation of cyclin inhibitor, cyclin and Cdk machinery, and that this process appears to be p53-dependent.
Recommended Citation
Xie, Jianxun, "Involvement of transcription factors in cadmium-induced apoptosis and cell cycle arrest in rat kidney cells" (2005). Open Access Dissertations. Paper 2084.
https://digitalcommons.uri.edu/oa_diss/2084
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