Abstract
Cadmium (Cd), a human carcinogen that triggers p38 signaling cascade and induces p53 activation. However, the subcellular localization of these stress-activated molecules remains to be elucidated. In this study, we adopted indirect immunohistochemistry and confocal microscopy techniques to illustrate how does Cd interfere with cellular morphology and the subcellular distribution of these stress-activated molecules in human diploid fibroblasts HFW and non-small cell lung adenocarcinoma cells CL3. Exposing cells to CdCl2 could induce apoptosis, nuclear morphological change and chromosomal abnormality as well as cause damage in mitochondria, microtubules and actin filaments. SB202190, a specific p38 inhibitor significantly decreased the Cd-induced actin filament disruption, suggesting that the p38 signaling is involved in the abnormal cellular morphology caused by Cd. Using specific O2.- and -NO fluorescent probes dihydroethidium and 4,5-diaminofluorescin diacetate, respectively, we have observed that CdCl2 induced these free radicals in time- and dose-dependent manners in both CL3 and HFW cells. A -NO synthase NOS inhibitor, N-nitro-L-arginine-methyl ester (L-NAME) decreased the Cd-induced -NO generation, implying -NO synthase is involved. Confocal microscopy analyses of indirect immunohistochemical stains showed Cd increased the levels of p38, p53, p21, Mdm2, phospho-p53 (Ser15) and phospho-p38 proteins. Upon Cd exposure, p38 and phospho-p38 shuttled from cytosol to nucleus, which is consistent with the finding that p38 kinase regulates gene expression. The increased p53, p21 and Mdm2 localized in the nucleoplasm but not the nucleolus, whereas the majority of phospho-p53 (Ser15) localized in nucleolus. The result suggests that nucleolus phospho-p53 (Ser15) participates in the transactivation of downstream genes such as p21, mdm2, gadd45 and bax due to that the nucleolus is the place for gene transcription. Mitochondria play a key role in apoptosis. The results that p38, phospho-p38, p53 and phospho-p53 (Ser15) all localized in mitochondria after Cd treatment, suggest these proteins may trigger cytochrome c release from mitochondria to cytosol and subsequently induce apoptosis.英文摘要……………………………………………………………………Ⅱ壹、中英文對照及縮寫……………………………………………………1貳、文獻回顧一、鎘化物與疾病…………………………………………………………3二、鎘之吸收與干擾鋅、鈣之平衡………………………………………3三、鎘化物與癌病…………………………………………………………4四、鎘之細胞與基因毒性…………………………………………………4五、鎘與含氧自由基之關係………………………………………………5六、鎘與一氧化氮之關係…………………………………………………6七、p38蛋白激脢訊號傳遞訊號路徑………………………………………7八、p53訊號傳遞訊號路徑………………………………………………8九、p53與Mdm2負回饋調控………………………………………………9十、p53抑癌基因細胞內運輸與分佈……………………………………10十一、鎘對生物訊號傳遞與基因表達的影響……………………………11十二、結語…………………………………………………………………11參、前言……………………………………………………………………12肆、材料與方法一、材料、化學藥品………………………………………………………14二、細胞培養液與緩衝液的配製…………………………………………14三、染劑配製………………………………………………………………15四、細胞培養………………………………………………………………15五、細胞處理與細胞固定…………………………………………………16六、蛋白質免疫螢光染色…………………………………………………16七、肌動蛋白絲染色………………………………………………………17八、粒線體染色……………………………………………………………17九、一氧化氮自由基 (.NO) 標定染色…………………………………18十、超氧自由基 (O2.-) 標定染色………………………………………18十一、共軛焦顯微鏡之觀察………………………………………………18十二、影像分析……………………………………………………………19伍、結果……………………………………………………………………20陸、討論……………………………………………………………………26柒、參考文獻………………………………………………………………30捌、圖表……………………………………………………………………43