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  • 學位論文

抑制第三拓樸酶對ALT細胞之影響

The effects of inhibition of Topoisomerase III in ALT cells

指導教授 : 鄧述諄

摘要


端粒(Telomere)是真核生物染色體末端的特殊結構,能夠防止染色體的末端被細胞辨認為損傷的訊息。人類的體細胞缺乏端粒酵素活性(Telomerase),端粒會隨著細胞分裂的進行而縮短,並限制細胞分裂的次數,因此被認為是一種預防腫瘤形成的機制。雖然大多數的癌細胞(85~90%)都具有端粒酵素活性來提供自己無限的分裂能力,有部分癌細胞(10~15%)不具有端粒酵素活性,並依據其端粒延長方式而被命名為ALT細胞(Alternative Lengthening of Telomeres)。先前的研究指出第三拓樸異構酶(Topoisomerase III)會參與酵母菌以及人類端粒的延長,在ALT細胞中利用RNA干擾技術抑制TOP3α的表現,會偵測到端粒酵素活性的重新啟動,本實驗則延續之前的研究,將進一步釐清在ALT細胞中抑制TOP3α對ALT細胞的影響。實驗發現在三個獨立細胞株當中都可偵測到端粒酵素活性,其端粒酵素的蛋白質表現上升,而端粒長度也類似具有端粒酵素活性細胞的端粒。APBs(ALT-associated PML bodies)是ALT細胞的一個特徵,包含許多蛋白,包括PML、Nbs1以及TRF2。我們利用免疫螢光法偵測TOP3α被抑制的ALT細胞,發現其APBs是消失的,代表細胞內的ALT pathway受到影響。我們也發現RIF1以及RIF2基因對於依賴Top3p/Sgs1p的端粒重組機制的建立是必須的。TOP3在ALT pathway中扮演重要角色,而目前尚無藥物能夠抑制Top3p,因此我們也尋找能抑制Top3p的藥物,發現大腸桿菌Tn5轉位子的transposase(Tnp)能夠抑制酵母菌的Type II 重組機制,我們將繼續研究它是否會影響Top3p的活性。

關鍵字

癌症 端粒重組 第三拓樸酶

並列摘要


Telomeres are the special structures of eukaryotic chromosomal ends, which can prevent chromosomal ends being recognized as damaging signals. Due to the lack of telomerase activity, the telomeres of human somatic cells shorten with the progress of each cell division, limit the cell division potential and was regarded as an tumor-preventing mechanism. Although most of the cancer cells possess telomerase activity to provide themselves with limitless replication potential, some cancer cells do not express telomerase, and named after their telomere elongation pathway as ALT cells (Alternative Lengthening of Telomeres). Previous research indicated that Topoisomerase III participates in yeast and human telomere elongation, telomerase activity was detected in ALT cells whose TOP3α expression was inhibited with RNA interference. This research continue previous experimments and try to further understand the effect of inhibition of TOP3α expression in ALT cells. We detected reactivation of telomerase activity in three independent si-h TOP3α Saos-2 stable clones. In these stable clones, we also detected elevated level of telomerase protein expression and found their telomere pattern was similar to that of telomerase positive cells. APBs(ALT-associated PML bodies)is one of the characteristics of ALT cells, including many protein such as PML, Nbs1and TRF2. We use Immunoflurescence assay to detect the APBs of Saos-2-TOP3αi cells and found they were diminished, this indicate the ALT pathway in these cells were inhibited. We also found the RIF1 and RIF2 genes are required for the establishment of Top3p/Sgs1p- dependent telomere recombination mechanism. TOP3 plays an important role in ALT pathway, so far there is no drug developed to inhibit Top3p, so we are looking forward to find Tpo3p-inhibiting drugs and found that the E. coli Tn5 transposase(Tnp)can inhibit Type II recombination mechanism of yeast, we will continue to investigate whether it can inhibit Top3p activity

參考文獻


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