MCF-7細胞是一種普遍的乳癌細胞株，他可因雌激素的刺激而使雌激素受體α和β表現，進而調節基因的轉錄以及細胞增生，然而，荷爾蒙活化的機制仍存在著爭議。我們遂結合化學修飾之探針與蛋白質體學的方法探討MCF-7細胞中由雌激素受體調控的轉錄路徑。利用雌激素應答序列修飾之金奈米粒子探針親和性純化經由具雌激素受體α有專一性的促進劑PPT處理3小時和未處理的MCF-7細胞。實驗結果顯示，液相層析質譜儀鑑定到雌激素受體α和許多轉錄調節因子像是輔助型蛋白、抑制型蛋白、組蛋白去乙醯酶 (HDAC)、熱休克蛋白 (HSP)以及參與在染色體重塑的SWI/SNF蛋白質。基於轉錄路徑分析，發現有許多我們鑑定到的且受到PPT刺激後有顯著變化的蛋白質皆以雌激素受體α和cMyc為中心，形成一個雙中心的作用網絡。我們以PPT誘發不同時間點 (0、0.5、3和24 小時)的實驗，探討雌激素受體α和cMyc的表現以及與雌激素應答序列鍵結的變化。我們更進一步探討雌激素受體α上S118的磷酸化程度，並發現S118的磷酸化會影響基因的轉錄。由西方墨點法的結果得知，PPT的誘發可快速在30分鐘內增加雌激素受體α的表現，同時亦使雌激素受體α上S118的磷酸化程度增加進而引導雌激素受體α入核並與雌激素應答序列鍵結；30分鐘之後，雌激素受體α的表現及其磷酸化的程度以及與雌激素應答序列的鍵結便開始下降，直至24小時，其程度便低於原本狀態。然而，我們發現PPT的誘發使cMyc的表現在3小時後有最明顯的增加。我們也發現cMyc在PPT誘發30分鐘時便開始與雌激素應答序列鍵結，而到了3小時達到最高峰，此時雌激素受體α的鍵結卻開始下降，暗示著cMyc藉由雌激素受體α與雌激素應答序列鍵結，但卻擔任使雌激素應答序列-雌激素受體α鍵結游離的角色，這樣的結論與體外的雌激素應答序列鍵結的實驗相符。此外，雌激素應答序列的冷光實驗也指出cMyc會抑制雌激素應答序列的轉錄。總結，所有的實驗結果指出cMyc扮演著共調節者的角色─抑制雌激素應答序列-雌激素受體α的鍵結以清除雌激素應答序列的複合體或在動態轉錄週期的後面階段扮演輔助型蛋白的角色以減緩轉錄的過程。

MCF-7 cell, a common breast cancer cell line, expresses estrogen receptor α and β (ERα and ERβ) which regulate gene transcription and cell proliferation but the ligand activation mechanism remains controversial. We conducted a chemical probe coupled proteomics approach to study ER-mediated transcriptional signaling in MCF-7 cells. Using estrogen response element (ERE) functionalized gold nanoparticle (AuNP-ERE) probe for affinity purification from MCF-7 cells under the condition with and without the 3-hour treatment of propyl pyrazole triol (PPT, 10-6 M) which is a specific agonist for ERα. Results indicated that ERα and many transcriptional regulators such as co-activators, co-repressor, histon deacetylases (HDAC), heat shock proteins (HSP) and SWI/SNF proteins involved in chromatin remodeling were identified by liquid chromatography-mass spectrometry (LC-MS) . Based on pathway analyses, ERα and cMyc were found to be two centers of the interaction map constructed by the identified proteins which exhibit significant changes upon PPT treatment. The time course (0, 0.5, 3, and 24 hours) study was then conducted to investigate how ERα and cMyc expression and their binding with ERE vary with PPT treatment. The change of the phosphorylation level on S118 of ERα was also investigated since it is known to affect gene transcription. Using immunoblot method, we showed PPT quickly increased the expression and S118 phosphorylation level of ERα and induced its nuclear translocation and ERE binding in 30 min. Beyond 30 min, the expression and phosphorylation level as well as ERE binding of ERα were decreased to a level below its original state after 24 hours. However, we found PPT increased cMyc expression at a later time: a peak maxima was observed after 3 hours of PPT treatment. We also found cMyc started to bind ERE at 30 min but reached a maxima at 3 hour under which the bound ERα was decreased, implying that cMyc was recruited to ERE through ER but acted to dissociate the ERE-ERα binding. This is in agreement with the in-vitro ERE binding assay. Furthermore, ERE luciferase assay indicated that cMyc represses ERE transcription. Taken together, our results indicate that cMyc acts as a co-regulator that suppresses the binding of ER or coactivators during the later phase of the dynamic transcriptional cycle to clear up the ERE complexes and slow down the transcription.

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