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題名 | Overexpression of Damaged-DNA-Binding Protein 2 (DDB2) Potentiates UV Resistance in Hamster V79 Cells=受損DNA結合蛋白DDB2的表現可增強倉鼠V79細胞抗紫外輻射 |
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作者姓名(中文) | 孫念康; 呂興邦; 趙清貴; | 書刊名 | 長庚醫學 |
卷期 | 25:11 2002.11[民91.11] |
頁次 | 頁723-733 |
分類號 | 415.132 |
關鍵詞 | 細胞凋亡; 受損DNA結合蛋白2; DNA修補; 抵抗性; 紫外輻射; Apoptosis; DDB2; DNA repair; Resistance; Ultraviolet radiation; |
語文 | 英文(English) |
中文摘要 | 背景: 細胞中具有紫外輻射損傷DNA的結合活性主要包含兩種蛋白:受損DNA結合蛋白1(DDB1)和2(DDB2),雖然還不清楚損DNA結合活性在細胞中之功能,但是已知DDB2發生突變會導致細胞對多種基因毒性物質的敏感性增加;其中包括紫外輻射,因此可推測DDB2在紫外輻射引起的DNA修補機制中扮演一定的角色。但是實際上有關DDB2參與DNA修補與對紫外輻射敏感性的證據仍舊缺乏。 方法:為了進一步研究DDB2的功能,我們建立一株可表現DDB2蛋白的倉鼠細胞株V79ddb2。在細胞經紫外輻射處理後,利用抗體檢測基因體上留存的光化合物;以及將帶有報導基因(CAT)的質體DNA,在細胞外經紫外輻射處理後送人細胞內,藉由其報導基因的表現,得以分析細胞修補受損DNA的效率。利用呈色分析(MTT)的方法檢測紫外輻射對細胞毒殺的敏感性,以及在顯微鏡下用螢光染色法(DPAPI)計算因紫外輻射引起染色體發生變化的凋亡細胞。 結果:我們發現該細胞株在12小時內可移除近50%的紫外輻射損傷的DNA;但是缺乏DDB2表現的母細胞株卻不具有這樣的修補能力。然而利用CAT基因當作報導基因的修補分析,顯示兩株細胞對紫外輻射損傷DNA的修補並無差異,此結果可推測DDB2可能只參與基因體之修補。另外在紫外輻射引起細胞凋亡與細胞毒性的分析方面,V79ddb2細胞也顯示比母細胞株有抗紫外輻射的能力。 結論:實驗結果顯示當V79細胞表現DDB2蛋白可提升細胞DNA修補能力以及降低紫外輻射對細胞的毒殺。因此推測DDB2可參與細胞抗紫外輻射的作用。 |
英文摘要 | Background: Ultraviolet radiation (UV) damaged-DNA binding (DDB) activity comprises two major components: damaged-DNA binding protein 1 (DDB1) and 2 (DDB2). Although the function of DDB is unclear, mutation on DDB2 is associated with cellular sensitivity to a variety of genotoxic agents including UV. It has been suggested that DDB2 may play a role in UV-induced DNA repair. However, evidence that DDB2 involves in DNA repair and UV sensitivity is lacking. Methods: To examine the role of DDB2, we established DDB2-overexpressing hamster V79 cell lines, V79ddb2, by stable transfection with full-length open reading frame of human ddb2 cDNA. Cells were irradiated with UV and determined its DNA repair activity by testing the remaining photoproducts on the chromatin and measuring the plasmid reactivation, respectively. UV induced cytotoxicity was determined by the colorimetric assay (MTT assay), and apoptotic cells exhibiting morphological features of chromatin condensation and nuclear fragmentation were counted after 4-diamidino-2-phenylindole (DAPI) staining. Result: DDB activity was increased in DDB2-overexpressing cell lines. Analysis on DNA repair indicated that UV photoproducts were removed in a time-dependent manner and there was greater than 50% of damage removed within 12 h in DDB2-overexpressing cells. In contrast, nearly all the damage remained unrepaired in V79 cells. However, using bacterial CAT gene as a reporter, both V79 and V79ddb2 cells demonstrated no difference in the reactivation of plasmid DNA carrying UV damage. These results suggest that DDB2 may involve in repair of bulky genomic DNA damage. Although a maximum of only 30% of apoptosis was induced, UV irradiation caused a dose-dependent apoptosis and cytotoxicity in these cell lines. V79ddb2 cells displayed resistance to UV-induced apoptosis and cytotoxicity. Conclusion: Our findings indicate that overexpression of DDB2 in V79 cell potentiates DNA repair and protects cells from UV-induced cytotoxicity. These results also suggest that DDB2 may be involved in the development of UV resistance. |
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