2026-11-092026-11-09https://scholars.csmu.edu.tw/handle/123456789/38070The chromosomal ends are capped with tandem copies of a simple DNA sequence (TTAGGG) termed the telomeric repeat. The telomere repeat binding protein 2 (TRF2) are specialized DNA-binding protein that are exclusively found at the telomere. Telomeric DNA and its associated proteins prevent illegitimate recombination, establishing a stable linearized chromosomal ends. They also perform chromosome positioning and segregation functions. Loss of TRF2 can induce apoptosis. Overexpression of functional TRF2 can overcome the senescence-inducing signals from critically short telomeres and thus allow cell growth in the presence. Results from recent studies of DNA damage from etoposide or radiation transiently increases TRF2 expression, might act as anti-apototic mechanisms in the DNA-damage response of malignant cells. In addition, TRF2 gene was observed in 83 ？Mof the 78 Taiwan non-small cell lung cancer specimen (Hsu C.P. et al., 2003). Therefore, TRF2 was high expression in lung cancer. Even if malignant cells which expose anticancer could increases TRF2 expression, the regulative mechanisms is unclear. To identify such mechanisms, we developed a series approach. Real time PCR detect TRF2 mRNA increases when lung cancer cell lines are treated with the anticancer drug-cisplatin and etoposide. Transient transfection of TRF2 promoter-reporter gene construct in the NSCLC cell lines (H1299 cells) will exhibit that anticancer drug induce the transcription of TRF2 promoter driven luciferase gene. Analysis of serial deletion mutations of hTRF-2 promoter will reveal which site of the Sp1, AP-2 and CREB response elements are located. Electrophoretic mobility shift assay will demonstrate that binding activity of transcriptional factor to the response element in the TRF2 promoter. We will analyze the mechanisms by which multiple regulatory factors, functionally interact to tightly maintain the inducible state of the TRF2 promoter in mortal cells. In the present study, we will characterize a response element in the human TRF2 promoter by anticancer drug. We will investigate the expression of TRF2 promoter by anticancer drug in lung cancer correlated with clinicopathological parameters.染色體終端序列(telomere)位於染色體兩端，由簡單重複核苷酸序列(TTAGGG)所組成，telomere repeat binding factor 2 (TRF2)，主要穩定染色體的完整性，避免與其他染色體發生融合或重組現象而引起染色體結構改變。當失去TRF2保護時，會引發細胞凋亡反應，而過量表現TRF2蛋白，會延緩細胞進入衰老階段。最近文獻指出血癌細胞株受到抗癌藥物etoposide 或放射線刺激下會短暫誘發TRF2 的表現，更穩定保護染色體的完整性，其表現可視為癌細胞受到DNA-damage反應下所產生的抗細胞凋亡機制 (Nakagami Y et al ., 2002 and Klapper W et al., 2003)。此外，在79位台灣非小細胞肺癌病人組織中發現TRF2高達83.3﹪表現量(Hsu C.P. et al., 2003)，因此可知TRF2在腫瘤組織是高度表現。雖然癌細胞受到抗癌藥物作用下會誘發TRF2的表現，但其作用機制不明，本計畫主要探討抗癌藥物如何調控TRF2表現，且研究抗癌藥物是經何種途徑誘導TRF2產生。或是抗癌藥物間接透過轉錄因子活化，進而結合到TRF2 promoter區位，所以本實驗擬先以定量PCR方式分析抗癌藥物-cisplatin和etoposide處理肺癌細胞是否會誘導 TRF2 增加。接著，將構築得自肺癌細胞的TRF2基因的上游約 700 鹼基對序列啟動子接合於螢光報告基因，其缺失突變變異株的質體，將其轉染至肺癌細胞中，藉由分析抗癌藥物處理過不同突變變異質體轉染過的細胞中luciferase活性，來了解抗癌藥物與TRF2 promoter調控關係，並知道其promoter區位的序列何者重要。並擬進一步分析抗癌藥物是否經由透過這些轉錄因子而影響TRF基因的表現，所以擬添加這些因子或使用此轉錄蛋白專一性的抑制劑至已轉染 TRF2 啟動子的肺癌細胞株，分析其轉錄活化能力。以瞭解cisplatin對TRF-2的調控機轉及其細胞毒性之關係。TRFtelomerasecisplatinProject