2026-11-092026-11-09https://scholars.csmu.edu.tw/handle/123456789/38551Telomerase expression is the hallmark of tumor cells in which this ribonucleoprotein complex activation may therefore be a rate-limiting or critical step in cellular immortalization and oncogenesis. In our previous study, we found that lung cancer patients without telomerase activity survive significantly longer than those with telomerase activity. Telomerase activity is an important prognostic factor in lung cancer patients. We have shown that reFIP-gts suppresses telomerase activity and inhibits transcriptional regulation of hTERT via a c-myc-responsive element-dependent mechanism. Here, we proved that reFIP-gts entered A549 lung cancer cell and localized in endoplasmic reticulum (ER). Accumulation of reFIP-gts in ER results in releasing ER stress marker, such as GADD153 and GRP78. Interestingly, treatment of reFIP-gts resulted in exporting hTERT to cytoplasm in A549 cells. Although we previously showed that treatment of reFIP-gts could reduce transcription of hTERT, our recent data showed that treatment of reFIP-gts might provide additional regulation of hTERT at post-tranlsational level. Therefore, this three years grant proposal aims to study whether treatment of reFIP-gts would cause cytoplasimc translocation of hTERT via mechanism(s) involved in ER stress. Therefore, in the first year study, we will focus on studying whether calcium release would be increased due to reFIP-gts mediated ER stress in A549 cells, and whether reFIP-gts suppresses telomerase activity via calcium-dependent and posttranslational mechanisms. In the second year study, we will use RNA interference and gene overexpression technique to study the effect of known ER stress markers, CHOP/GADD153 and GRP78, on telomerase activity. In addition, ER stress related gene chip will be used to compare gene expression patterns among cells treated with reFIP-gts, with Thapsigargin (ER stress inducer), and without any treatments. If any candidate genes were identified, RNAi and gene overexpression will be applied on these genes for studying the involvement of these genes on telomerase activity. Whether ER stress participated in reFIP-gts suppresse telomerase activity. In the third year study, we will establish animal model to confirm that reFIP-gts have anti-telomerase ability. This novel finding may provide useful information for the therapeutic strategy in cancer therapy.染色體端粒.的再活化促使正常細胞擁有高度的細胞增殖速率和無限次的複製能力，渡過 衰老死亡的危機期進而走向不朽化並提高正常細胞轉型成惡性腫瘤的機率，是癌化過程中必 備的條件。本研究室與其他學者皆曾發現當肺癌患者的腫瘤組織中若喪失端粒.活性其存活 率較高，說明端粒.活性的表現強弱可作為肺癌病人預後的指標。先前我們已初步證實重組 松杉靈芝免疫調節蛋白(reFIP-gts)透過抑制c-Myc 結合到hTERT 啟動子，進而抑制A549 肺癌細胞端粒.活性。為了要釐清reFIP-gts 是否進入細胞核直接影響或聚集在細胞質而間 接影響端粒.的調控，我們以細胞免疫染色法觀察到reFIP-gts 進入A549 細胞後，累積在 內質網但不會進入細胞核。當大量的蛋白或某些病毒感染易累積至內質網容易造成內質網壓 力並誘發內質網釋出鈣離子，進而誘發其訊息傳遞，促使細胞凋亡。有鑑於此，我們擬分析 reFIP-gts 是否透過內質網壓力的誘發抑制肺癌細胞的端粒.活性，並探討其相關機轉。本 研究將分三年來完成，第一年探討reFIP-gts 對於內質網壓力的產生是否伴隨著細胞內鈣離 子增加，鈣離子所誘發的訊息傳遞是否會影響端粒.轉譯後修飾的表現進而改變端粒.在細 胞內的穩定度及蛋白在細胞座落位置的改變。第二年探討reFIP-gts 內質網壓力的誘發是否 與端粒.活性抑制有關，以內質網壓力的誘導物Thapsigargin 作為正對照組分析是否也會抑 制端粒.活性。而GADD153 和GRP78 是內質網壓力最具代表性的指標基因之ㄧ，因此將 建立過量表現及RNA 干擾技術降低GADD153 和GRP78 基因的A549 細胞株來確認內質網 壓力是否會抑制端粒.活性。倘若不是透過GADD153 和GRP78 基因所調控，我們將利用 內質網壓力相關基因的基因晶片比較reFIP-gts 和Thapsigargin 誘發後的相關基因表現，再 將這些相關基因利用上述相同策略來更進一步證實內質網壓力抑制端粒.活性的可信度，最 後分析reFIP-gts 是否能有效抑制其他肺癌細胞株端粒.活性，若無法抑制端粒.活性是否 與內質網壓力誘發的差異有關。第三年探討動物模式來確認內質網壓力的誘導物 Thapsigargin 和reFIP-gts 是否能抑制A549 細胞所產生腫瘤，並分析腫瘤組織內端粒序列長 度縮短和端粒末端融合來證明是reFIP-gts 抑制腫瘤細胞端粒.活性所造成，同時也與內質 網壓力調節端粒.活性相關的基因將過量或RNA 干擾表現的A549 細胞注入裸鼠分析是否 會影響腫瘤的生長。松杉靈芝免疫調節蛋白質端粒內質網壓力鈣離子轉譯後修飾reFIP-gtsTelomeraseER stressCalcium releasePosttranslational modificationProject