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  • 學位論文

探討非小細胞肺癌之人類終端反轉錄酶的啓動子甲基化及組織蛋白乙醯化狀態對終端酶活性之影響

The influence of hTERT promoter methylation and histone core acetylation status to telomerase activity in non-small cell lung cancers

指導教授 : 柯俊良

摘要


人類染色體終端 (Telomere) 是一段重覆的DNA序列 (5’-TTAGGG-3’),它可以保護染色體不被分解而失去人體所必需的基因,同時它也是細胞持續分裂時所必需之結構。目前的研究報告指出,在人類正常體細胞中,其染色體終端會隨著每次細胞分裂而縮短,同時其終端酶的活性也是較低的;相反地,在癌化細胞中,其不但具有完整且較短的染色體終端,其終端酶活性也會上升。因此,終端酶活性的表現可能與細胞癌化的過程有關。 另一方面,人類DNA的甲基化程度及組織蛋白乙醯化狀態已知是會影響基因表現的主要機制。所以本論文主在探討終端酶活性和人類終端酶反轉錄酶的啟動子之甲基化和乙醯化狀態之間的關係,同時,也從非小細胞肺癌病患的病理組織細胞中來探討此一關係。 首先我們利用去甲基化藥劑 (5-Aza-2’-deoxycytidine: 5-aza-dC) 及組織蛋白去乙醯酶抑制劑 (Trichostatin A: TSA) 來分別處理兩株人類肺癌細胞株 (A549, H1299)。我們發現去甲基化藥劑 (5-aza-dC) 會促進H1299及A549細胞中的人類終端酶反轉錄酶 (hTERT) 的表現,而組織蛋白去乙醯酶抑制劑 (TSA) 則會抑制肺癌細胞 (H1299和A549) 中hTERT的表現,而這種抑制作用主要是經由減低轉錄因子 (c-Myc和Ets-2) 與hTERT promoter的鍵結能力而進一步抑制肺癌細胞 (H1299和A549) 中終端酶的活性表現。 接下來,我們收集了62位非小細胞肺癌病患之腫瘤組織及鄰近正常肺部組織來探討人類終端酶反轉錄酶的啟動子之甲基化和終端酶活性之相關性。結果我們發現不論在腫瘤組織中或鄰近正常肺部組織中當hTERT沒有表現時,其人類終端酶反轉錄酶的啟動子具有高度甲基化的情形較明顯(p=0.029 and p=0.01)。同時我們也觀察到染色體終端在腫瘤組織細胞中是較短的,但是卻無足夠的證據來証實人類終端酶反轉錄酶啟動子甲基化和染色體端粒酶活性之間的相關性。 經由這兩項實驗,我們發現反轉錄酶的啟動子的甲基化狀態的確會影響終端酶活性之表現,然而卻無法証實會進一步影響到病患的疾病預後。

並列摘要


Telomeres consist of tandem oligonucleotide repeats (5’-TTAGGG-3’) that cap the ends of eukaryotic chromosomes to prevent further degradation and loss of human essential gene. Functional Telomeres are also essential for continued cell proliferation. By present research report, telomeres progressively shorten during each cell division without increasing the telomerase activity in most human cell. However, tumor cell generally have functional and short telomere lengths and revealed upregulated telomerase activity. Therefore, telomerase activity is hallmarks of tumorgenesis. On the other hand, DNA methylation and histone acetylation are important mechanism of epigenetic regulation without affecting the DNA sequence. Our in vitro and in vivo experiences tried to prove the relationship between telomerase activity and the methylation or histone acetylation of promoter region in human telomerase reverse transcriptase (hTERT). 5-Aza-2’-deoxycytidine (5-aza-dC) and Trichostatin A (TSA) are both added into the culture medium of two human non-small cell lung cancer cell line (H1299 and A549). Demethylating agent (5-aza-dC) activated the hTERT mRNA expression in H1299 and A549 cell lines. However, the TSA repressed the hTERT mRNA expression and telomerase activity in H1299 and A549 cell lines. TSA targets c-Myc and Ets-2 binding sites within the core region of the hTERT promoter to suppress the telomerase activity of H1299 and A549 cell lines. Genomic DNAs were extracted from non-small cell lung cancer samples and adjacent normal lung tissue of 62 patients. Hypermethylation status of the promoter of hTERT was found in low expressed hTERT of tumor sample and adjacent normal lung tissue (p=0.029 and p=0.01). Extremely shortened telomere length in tumor sample than adjacent normal lung tissue without correlated with the methylation status of promoter of hTERT and telomerase activity also noted in our experience. Base on these two experiences, the data show that the methylation and histone acetylation status in core promoter of hTERT could control the expression of hTERT and further telomerase activity. However, we can’t demonstrate that they could be potential biologic marker targets for clinical outcome of non-small cell lung cancer patients.

並列關鍵字

telomere hTERT telomerase activity

參考文獻


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