以太平洋紫杉醇引發之 hTERT 表現量下降探討其CTCF 結合區上 DNA 甲基化之調控機制

Abstract

人類端粒逆轉錄酶 (human telomerase reverse transcriptase, hTERT) 為端粒酶之主要催化單位。在正常體細胞中並不會表達，但會在大部分腫瘤及幹細胞中表現。絕大多數正常體細胞及癌細胞間 hTERT 的表現與否，其轉錄遏阻因子 CTCF (CCCTC-binding factor) 結合區的甲基化程度扮演著關鍵性的角色。本實驗室在先前研究中發現在光動力致壓效應下，可觀察到 hTERT 基因表現降低的原因是透過將 CTCF 結合區去甲基化所造成。過去文獻指出抗癌藥物，如太平洋紫杉醇 (Taxol) 等具有抑制癌細胞 hTERT 基因表現量下降之能力，然作用機制仍不清楚。故本研究主要目的為探討 Taxol 處理後胞內 hTERT 基因表現量降低現象是否與其甲基化程度相關，並期望因此對 hTERT 基因調控機制有更進一步的了解。因此本研究首先以 Taxol 處理癌細胞後，觀察到 hTERT 表現量確實有下降現象，而相關的去甲基化因子 growth arrest and DNA damage-inducible 45α (GADD45α) 及 inhibitor of growth 1 protein (ING1) 表現量則有增加之情形。進一步以亞硫酸氫鹽-基因組測序法 (Bisulfite genomic sequencing, BGS) 證明以 Taxol 處理後會使 CTCF 結合區之甲基化程度降低。最後利用染色質免疫沉澱 (chromatin immunoprecipitation, ChIP) 證明以 Taxol 處理後，GADD45α 及 ING1 蛋白質會鍵結至 CTCF 結合區進行去甲基化。這些結果顯示 hTERT 基因在受到 Taxol 藥物處理後，會促使 GADD45α 及 ING1 蛋白對 CTCF 結合區進行去甲基化，進而使轉錄遏阻因子 CTCF 可順利結合 CTCF 結合區抑制 hTERT 基因轉錄。此外，我們亦使用 Doxorubicin 等藥物處理癌細胞，確認此調控機制並非 Taxol 專一性。因此，我們推論此調控機制可能是細胞在遭受壓力環境之下普遍存在之現象。

Human telomerase reverse transcriptase (hTERT), the catalytic subunit of telomerase, is expressed in most immortalized and cancer cells but not in normal somatic cells. The expression of hTERT has been reported to be associated with the DNA methylation status in its repressor, CCCTC-binding factor (CTCF), proximal exonic binding site. Previously, we have found that oxidative stress induced by photodynamic treatment could reduce the hTERT expression, which resulted from the reducing of DNA methylation on the CTCF binding site. The reduced expression of hTERT was also found in cells treated with Taxol. However, the regulatory mechanism is still unclear. The aim of this study is to investigate whether the reduction of hTERT gene expression in Taxol treated cells relates to the methylation status of hTERT gene. We found that the hTERT gene is down-regulated and the DNA demethylation related proteins, the growth arrest and DNA damage-inducible 45α (GADD45α) and the inhibitor of growth 1 (ING1) protein, are up-regulated in Taxol-treated cancer cells. Using bisulfite genomic sequencing, we found that hypomethylation on the CTCF binding site of hTERT might be responsible for its suppressed down-regulation. Finally, the data of chromatin immunoprecipitation (ChIP) assay demonstrated that GADD45α and ING1 protein indeed bind to the CTCF binding site and promote the DNA demethylation in cells treated with Taxol. Similar results were also found in cells treated with other chemotherapeutic drug, such as doxorubicin. Taken together, we found that GADD45α and ING1 mediated DNA demethylation of CTCF binding site of hTERT gene may regulate the expression of hTERT under Taxol treatment via recruiting the transcriptional repressor CTCF to the proximal exonic region of hTERT. Telomerase activation plays a critical role in human carcinogenesis through the maintenance of telomeres. The regulatory mechanism of hTERT found in this study might offer a new therapeutic strategy of cancers.