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Title: | 探討第二型拓樸異構酶參與在致癌性重金屬鎘誘發之DNA斷裂及突變的角色與機轉 The involvement of topoisomerase II in the carcinogenic cadmium-induced DNA breakage and mutagenesis |
Authors: | Wei-Jer Chang 張維哲 |
Advisor: | 李財坤 |
Keyword: | DNA拓樸異構酶,鎘,致癌,DNA損傷,黑色素瘤, DNA topoisomerase,Cadmium,Carcinogenesis,DNA damage,Melanoma, |
Publication Year : | 2011 |
Degree: | 碩士 |
Abstract: | 鎘(Cd2+),是一種有細胞毒性且具致癌性的金屬。和其他的致癌性金屬一樣,鎘已經在許多研究中被證實會造成細胞的轉型和誘發腫瘤生成,但對於它致癌的分子機轉仍然有待了解。在我們實驗室之前的研究中發現,DNA拓樸異構酶(DNA topoisomerase, TOP)可能在鎘所造成的DNA斷裂中扮演重要的角色,而第二型拓樸異構酶(TOP2)所造成的DNA斷裂已經知道會促進DNA突變而加速癌症的發生,所以我們利用細胞培養系統與動物實驗來探討TOP2是否參與鎘的致癌機轉以及可能的作用機制。有趣的是,我們發現在經過鎘處理後的細胞中,有TOP2可切性複合體(TOP2 cleavable complex, TOP2cc)的形成;利用RNA干擾技術(RNA interference, RNAi)分別降低TOP2的同功酶TOP2α和TOP2β的表現,也發現鎘所造成的DNA斷裂與細胞毒性在缺乏TOP2β的細胞中都有較顯著地降低,而在TOP2α缺失的細胞則沒有明顯的差異,這意味著鎘可能偏好作用於TOP2同功酶之一的TOP2β上。此外,鎘所引發之DNA重組(DNA recombination)在缺乏TOP2β的細胞中亦有顯著地下降,這也更加支持TOP2β在鎘的作用機制中可能扮演重要的角色。最後我們利用兩階段小鼠皮膚致癌模式系統(two-stage mouse skin carcinogenesis model)及TOP2β的皮膚條件性基因剔除小鼠(skin-specific conditional knockout mice)來探討TOP2β在鎘的致癌機轉中的重要性。在此實驗中我們發現了和野生型(wild-type)的小鼠相比,鎘所誘發的黑色素癅(melanoma)在TOP2β的基因剔除鼠(TOP2β-)上有明顯地減少,這指出在鎘的致癌機轉中TOP2β是有參與的。因此,以上的實驗結果證實了鎘可能經由創造TOP2βcc產生DNA斷裂,進而引發DNA重組的發生,最終導致腫瘤的生成。 Cadmium (Cd2+), a toxic metal, has many industrial and daily applications. Like other carcinogenic metals, it has been not only reported to be cytotoxic, but also classified as a carcinogen. However, Cd2+ is not directly genotoxic and mutagenic in the bacterial system and is only weakly mutagenic in mammalian cells. Thus, indirect mechanisms have often been implicated in the Cd2+ carcinogenicity. In our previous study, we have found that topoisomerase II (TOP2) might be involved in the Cd2+-induced DNA breakage. Interestingly, TOP2-mediated DNA breakage has also been reported to be mutagenic and carcinogenic. In my proposal, we sought to determine the potential role of TOP2 in the Cd2+-induced DNA breakage, mutagenesis and tumor formation. We have found that Cd2+ could induce formation of TOP2 cleavable complex (TOP2cc). Both DNA breaks and cytotoxicity are reduced in shTOP2β cells, indicating that Cd2+ might target TOP2β preferentially. Cd2+-induced plasmid integration is also reduced in shTOP2β cells, suggesting an important role of TOP2β in Cd2+-induced DNA sequence rearrangements. To examine the role of TOP2β in Cd2+-induced carcinogenesis, a two-stage mouse skin carcinogenesis model was used. In this model, we found that the incidence of Cd2+-induced melanoma on the skin of drug-treated mice is significantly higher in wild-type than in skin-specific top2β-knockout mice. This data thus suggest that Cd2+ carcinogenesis is, at least in part, TOP2β dependent. Overall, these results indicate that Cd2+ induces TOP2β-mediated DNA damage and DNA sequence rearrangements, resulting in Cd2+ carcinogenesis. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/34240 |
Fulltext Rights: | 有償授權 |
Appears in Collections: | 微生物學科所 |
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