中文摘要

人類的WWOX基因位於第16對染色體長臂23.3-24.1的位置上， 它所轉譯出的WOX1蛋白亦被命名為WWOX或FOR被定義為腫瘤抑制蛋白，以及細胞凋亡前蛋白。 完整的WOX1含有兩個胺基端的WW區域，一個細胞核結合區 nuclear localization sequence (NLS)，和一個位於碳基端的短鏈乙醇脫氫酵素區段 short-chain alcohol dehydrogenase/reductase (ADH/SDR)，為粒線體結合區。WOX1參與許多訊息傳遞的路徑，例如 in vitro細胞實驗證明，當細胞受到刺激時， WOX1第一個W區域Try33的位置會被磷酸化且進一步進入細胞核。動物模式的in vitro研究指出，WOX1的角色參與在神經發育和死亡，包括長期光照引發神經退化的WOX1表現改變。etoposide是一種能導致去氧核糖核酸的損傷和引發細胞的死亡的第二型拓樸異構酵素， 在臨床上被使用為化療的藥物，化學療法可能會造成神經學上的併發症和神經細胞的去氧核糖核酸的損傷，進而引發許多神經退化疾病的病理現象。 於此實驗中我們使用etoposide引起的神經細胞死亡來探討WOX1參與其中的角色，首先是利用MTT Assay來決定etoposide的劑量和給藥的時間點。經由去氧核糖核酸斷裂的實驗證實etoposide能使神經母細胞瘤細胞株引發細胞凋亡。而在核質分離實驗中， 細胞質中磷酸化WOX1 (p-WOX1) 的表現會隨著暴露於etoposide的時間增加之下而有減少的情形， 而細胞核中磷酸化WOX1的表現則會在etoposide處置下兩小時達高峰。 細胞死亡標定物caspase-activated DNase (CAD)的表現無論是細胞核或細胞質中都於etoposide處置下6小時後隨著藥物暴露時間的增加而增加表現。此外WOX1與轉譯因子activation transcription factor 3 (ATF3)有共同表現的現象。 實驗中初步認為磷酸化WOX1 (p-WOX1)可能在etoposide的暴露之下能促進細胞凋亡,而轉譯因子ATF3可能也參與及促使WOX1大量表現來調控細胞凋亡，這部分值得未來更近一步討論，目前的實驗結果可以進一步提供我們利用基因轉殖的方法來探討etoposide引起的神經細胞死亡中WOX1參與的角色。

ABSTRACT
Human WWOX gene which located on ch16q23.3-24.1 encodes a putative tumor suppressor WOX1; also known as WWOX or FOR. The full-length of WOX1 processes two N-terminal WW domains, a nuclear localization sequence (NLS), and a C-terminal short-chain ADH or SDR domain, containing a mitochondrial binding site. WOX1 participates in multiple signaling pathways, including apoptosis. Upon various stimulations, WOX1 becomes activated via Tyr33 phosphorylation and nuclear translocation in vitro. The role of WOX1 in relative to the neuronal development and photoreceptor degeneration was reported recently. Etoposide, an inhibitor of topoisomerase II induces DNA damage and triggers cell death, is used as a chemotherapeutic agent. Chemotherapies incur neurological complications and DNA damage in neurons implicated in the pathogenesis of several neurodegenerative disorders. In this study, the role of WOX1 in etoposide induced neuronal death was investigated. The time course and dose of etoposide was determined by MTT assay. By DNA fragmentation assay, etoposide was found to induce apoptosis in neuroblastoma SH-SY5Y cells. The expression of p-WOX in cytosol was decreased along with increasing the exposure of time of etoposide, while the expression of phosphorylated WOX1 (p-WOX) in nucleus attained to the peak at 2 hours. In the meanwhile, the expression of caspase-activated DNase (CAD) was observed to increase at 6 hours after the treatment of etoposide in cytosol and nucleus and tended to be time dependent. The activation of WOX1 in related to immediated-early genes (IEGs) such as activation transcription factor 3 (ATF3) expressed and translocated to nuclei was also observed in this study. Taken together, those results suggested WOX1 activation might promote apoptosis and ATF3 might involved in the alternation of WOX1 in etoposide-induced neuronal death. The interaction of WOX1 and transcription factors requires investigating. Moreover, these experimental data might provide a thought for exploring the role of WOX1 in chemicals-induced neuronal death.

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