Cebpd在老鼠胚胎纖維母細胞中對Cisplatin反應的分子機轉探討

Characterization of Cebpd in response to cisplatin in mouse embryonic fibroblasts

單婉如(Wa-Ju Shan)

指導教授 ： 黃阿梅 老師

高雄醫學大學/醫學院/生物化學研究所/碩士(2007年)

摘要

C/EBP(CCAAT/enhancer binding protein)屬於basic leucine zipper(bZip)轉錄因子，有六個主要成員有:α，β，δ，γ，ε，ζ。所有的C/EBP蛋白在C端都含有一段保守leucine-rich的序列可以形成homo-或hetero-dimer，及basic region可與DNA結合，而N端則具有轉錄活化及抑制的調節區域。所有的C/EBP家族在不同的細胞或者組織中都存在著某些調節的角色，例如促進生長、調控細胞週期、促進細胞凋亡的功能等。Cebpd首先被發現為發炎反應的基因，但在目前也已知能參與在多種不同的生理功能。若將老鼠Cebpd基因剔除後，發現老鼠的發育正常及其特徵上無明顯差異，證實缺乏Cebpd並不會影響老鼠的生存。然而在老鼠的胚胎纖維母細胞(mouse embryonic fibroblasts ; MEFs)的研究中卻發現，Cebpd剔除的primary MEFs有明顯的染色體增生及重組的現象(Huang, et al. Oncogene 2004)，此實驗結果顯示Cebpd可能參與在維持細胞基因體的完整性並具有腫瘤抑制基因功能。
在本實驗中首先發現，剔除Cebpd的E1A-immortalized MEFs比WT細胞有較好的生長現象，此結果與先前之研究相符合。利用多種不同的抗癌藥物處理細胞時發現，Cebpd剔除的MEFs特別對cisplatin(CDDP)具有專一的抗性。CDDP常被利用作為臨床上的抗癌藥物，但處理CDDP常會伴隨著病人抗藥性的產生，其主要的分子機制仍未明。根據許多的研究顯示CDDP可能是透過活化p53以及MAPK (mitogen activated protein kinase) pathways而導致誘發下游的細胞死亡路徑。本實驗證實，剔除Cebpd 的MEFs細胞中p53及MAPK家族的路徑與Wild type具有不同的活性，顯示Cebpd可能參與在CDDP引起的相關反應，同時也有G1 checkpoint regulation 的缺失。因此未來可更進一步分析Cebpd參與在CDDP抗性上的分子機轉。

關鍵字

老鼠纖維母細胞

並列摘要

C/EBP(CCAAT/enhancer binding protein) family is a basic leucine zipper (bZip) transcription factor, and contains six members: α, β, δ, γ, ε and ζ。All C/EBP proteins share conserved C-terminal regions that contain leucine-rich dimerization motifs adjacent to basic DNA-binding regions. The N-terminal regions are more diverse and contain transcriptional activation and repression domains. Cebpd was first characterized as an acute phase inflammatory response gene. It has been implicated in diverse biological functions. Cebpd null mice are fertile and no overt phenotypes. However, Cebpd knockout mouse embryonic fibroblasts (MEFs) showed severe chromosomal rearrangement in vitro (Huang, et al. Oncogene 2004). This suggests that Cebpd is a potential tumor suppressor gene based on its role in maintenance of genomic integrity.
In this study, Cebpd knockout MEFs showed better proliferation than wild type. Among all tested anticancer drugs, the Cebpd knockout MEFs displayed significant resistance to cisplatin. Cisplatin is a wildly used anticancer drug. However, its clinical efficacy is largely limited to its toxic effects on normal cells and the development of drug resistance within tumor cells. It has been suggested that cisplatin activates p53 and MAPK pathways to induce apoptosis. The activation of p53 and MAPK pathways are altered in Cebpd knockout MEFs upon cisplatin treatment. This strongly suggests that Cebpd might contribute to the cisplatin-induced cellular responses and the control of G1 checkpoint regulation . The molecular mechanism by which Cebpd might involve will be further investigated. This would provide a better understanding of cisplatin resistance.

並列關鍵字

Cebpd ； CDDP ； MEFs

參考文獻

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