腫瘤抑制蛋白雙色胺酸功能區氧化還原酶(WW domain-containing oxidoreductase)又名WWOX、FOR或WOX1，在調節血癌T細胞生長及細胞凋亡的角色並未被全然瞭解。在這個研究中，我使用急性淋巴母細胞白血病細胞MOLT-4來探討WOX1在調節細胞生長中的角色。為了瞭解WOX1在血癌T細胞中參與的分子機制，從過去的實驗中，發現WOX1與NF-κB的抑制者，IκBα，存在著交互作用。當MOLT-4細胞受到Ionophore/PMA (IoP)的刺激後，我們發現在1至2小時內，WOX1在Tyr33的位點磷酸化程度下降，2至5小時內緊接著是ERK的磷酸化程度上升，而IκBα在3至15小時內會被降解，最後在15至24小時後，CD3及CD8的表現程度上升。我們更進一步發現IoP可以增加S及G2/M時期的細胞族群而促進細胞週期的進行。我們使用MEK1的抑制劑，U0126，阻止了CD3及CD8表現程度上升的現象，這個實驗結果說明了Raf/MEK/ERK這條訊息傳遞路徑可能參與這些蛋白質的表現調控，並且與IκBα及其可能結合的蛋白質有關。利用共軛焦螢光顯微鏡，我們發現部份WOX1在粒腺體內與IκBα有共位現象(Colocalization)，而IoP會造成這個共位現象在3小時內下降。同時利用共同免疫沈澱(Co-immunoprecipitation)，發現IoP造成WOX1及IκBα在2小時內分開，而Tyr33磷酸化的WOX1會與IκBα結合。利用共同免疫沈澱，WOX1藉由IκBα N端的ankyrin-repeat區域進行結合，而不是C端的PEST domain。總結上述結果，我們發現了一個新的WOX1結合蛋白，IκBα。而WOX1與IκBα間的結合可能調控MOLT-4 T細胞的生長。

The role of tumor suppressor WW domain-containing oxidoreductase, known as WWOX, FOR or WOX1, in regulating T leukemia cell proliferation and apoptosis is not well understood. In this study, acute lymphoblastic MOLT-4 leukemia T cells were used to investigate whether WOX1 participates in the cell growth regulation. To understand the molecular pathway of WOX1 signaling in T-cell leukemia, WOX1 was shown to interact with IκBα, an inhibitor of transcription factor NF-κB. Upon stimulation of MOLT-4 cells with ionophore/PMA (IoP), downregulation of WOX1 phosphoryation at Tyr33 occurred in 1-2 hrs, followed by ERK phosphorylation in 2-5 hrs, IκBα degradation in 3-15 hrs, and upregulation of CD3 and CD8 in 15-24 hrs. IoP promoted the cell cycle progression by increasing cell populations at the S and G2/M phases. MEK1 inhibitor U0126 blocked the expression of CD3 and CD8 proteins, suggesting the involvement of the Raf/MEK/ERK pathway and that IκBα binds candidate proteins in this pathway. By confocal microscopy, WOX1 colocalized, in part, with IκBα in the mitochondria, and IoP induced the dissociation in 3 hrs. In parallel, IoP induced WOX1 dissociation from IκBα in 2 hrs as determined by co-IP, in which Tyr33-phosphorylated WOX1 physically bound IκBα. Also, by co-IP, WOX1 bound IκBα via N-terminal ankyrin-repeat region but not the C-terminus PEST domain. Taken together, a novel WOX1 binding protein, IκBα, is revealed. And the interactions of WOX1 and IκBα may regulate cell growth and proliferation in MOLT-4 T-cell.

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