Cebpd蛋白在老鼠胚胎纖維母細胞中參與基因致毒物MMS作用之機轉探討

Characterization of genotoxin MMS-induced Cebpd protein in MEFs

賴彥任(Yan-Ren Lai)

指導教授 ： 黃阿梅

高雄醫學大學/醫學院/醫學研究所/碩士(2008年)

https://doi.org/10.6832/KMU.2008.00155

摘要

C/EBP 轉錄調控因子(CCAAT/Enhancer Binding Protein)是由Cebpa、Cebpb、Cebpg、Cebpd、Cebpe以及Cebpz六個成員所組成的。這些成員都具有basic leucine zipper以及與DNA的結合部位，可以分別形成Homo-或Hetero-dimer和DNA結合。其中Cebpd在許多不同種類的細胞中都會經由外來的刺激而表現，具調控細胞複製、分化、代謝、發炎反應以及維持染色體穩定等角色。本實驗室先前的研究發現，Cebpd缺失的老鼠胚胎纖維母細胞(Cebpd KO MEFs)透過改變p53和Erk活化的路徑提高對於cisplatin的抗性。因此推論Cebpd可能參與在調控細胞對於基因致毒物之反應。Methyl methanesulophonate (MMS)是一種DNA烷化劑，主要經由形成DNA adducts而對細胞造成損害，而細胞可利用base excision repair的方式來修補。本研究中首先發現MMS短時間高劑量的處理會造成MEFs細胞凋亡。而Cebpd KO MEFs (NKO)相較於WT MEFs (NWT)對MMS有較高的抗性。此結果可由型態及分析細胞凋亡相關的蛋白質印證。以Comet-NE assay分析MMS對NWT及NKO MEFs細胞造成的DNA受損情形，初步結果發現MMS皆可對兩種基因型細胞有高達約80%的DNA damage，顯示Cebpd參與的基因致毒物抗性應是在細胞DNA受損後之訊息傳遞路徑。MMS的刺激在Cebpd WT MEFs可誘導Cebpd、phospho-Ser-15 p53及p21蛋白質的量。有趣的是p53的mRNA沒有因為MMS的刺激而增加，而p21的mRNA表現量卻隨著MMS處理的時間而增加所以MMS可能是透過post-transcriptional的調控誘發Cebpd，p53和p21的活化。另外，由於p38的磷酸化主要會進一步磷酸化p53，而MMS的處理可以誘導p38的磷酸化，但是Cebpd的存在與否並不影響p38磷酸化的程度。所以推測Cebpd可能是在p38下游並且與p53有交互作用來調控細胞的行為。綜合以上的實驗結果，顯示Cebpd在細胞遭受基因致毒物時的反應上扮演重要的角色。未來可更進一步探討Cebpd參與在p53、p21之mRNA及蛋白質之可能調控和Cebpd參與在MMS所引起的DNA受損及修補的分子機制。

關鍵字

老鼠胚胎纖維母細胞 ； 基因致毒物

並列摘要

The transcription factors C/EBP (CCAAT/Enhancer Binding Protein) family is a highly conserved family of leucine zipper composed of six members: Cebpa, Cebpb, Cebpg, Cebpd, Cebpe and Cebpz. Cebpd originally was identified as an inflammatory response gene. Cebpd is expressed in a variety of tissues and cell types and involved in the control of cellular proliferation, differentiation, metabolism, inflammation and maintenance of genomic stability. In our previous studies, we had demonstrated that Cebpd knockout (KO) mouse embryonic fibroblasts (MEFs) exhibited significant higher resistance to cisplatin through altering the p53 and Erk signaling pathways. This suggests that Cebpd could serve as a mediator in response to genotoxic agents. Methyl methanesulophonate (MMS) is an alkylating agent. The removal of MMS-induced DNA adducts is mainly through base excision repair. In this study, we found that Cebpd KO MEFs (NKO) showed higher resistance to MMS when compared to wild type. In addition, Cebpd protein and p53 protein activation (phospho-Ser-15) can be induced by MMS within five hours in NWT. p21 protein, a well-known p53 target gene, was also induced at the same kinetics. However, Cebpd and p53 mRNA level were not induced after MMS treatment. The phosphorylation of p38 can also be induced by MMS, but there was no difference whether Cebpd existence or not. Phosphorylated p38 can directly activate p53. These data indicate that Cebpd may play an important role in regulating of apoptosis and DNA repair in MEFs. The molecular mechanism by which Cebpd might involve in MMS-induced DNA damage response needs to be explored. This study reveals a potential role of Cebpd in MMS-induced DNA damage response in MEFs.

並列關鍵字

Cebpd ； MMS ； genotoxin ； MEFs

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