探討betaⅡ-tubulin對於CBF1所媒介的Notch訊息路徑之調控

Study on the control of CBF1-dependent Notch signaling by betaⅡ-tubulin

蓋允芳(Yun-Fang Kai)

指導教授 ： 葉添順

臺北醫學大學/醫學院/細胞及分子生物研究所/碩士(2005年)

摘要

中文摘要 Notch訊息路徑其會參與細胞之增生、分化以及凋亡等許多發展過程。此外,許多文獻也指出一些人類疾病與Notch受體蛋白異常活化有相關。當位於細胞膜上Notch受體與鄰近細胞膜上的Notch接合體結合而被活化後,會引發Notch受體上的一連串切割反應,並在細胞內形成Notch受體蛋白細胞內區域( Notch IC ),接著轉運進入到細胞核內去與CSL蛋白以及一些其他細胞因子結合,進一步去調控標地基因的表現。然其中牽涉到的一些其他細胞因子對於Notch訊息傳遞路徑的調控,其相關機制卻仍待釐清與了解。本實驗室於先前已證實核內?Ⅱ-tubulin 蛋白與活化型Notch之間有相互結合。然而,對於核內?Ⅱ-tubulin如何出現在細胞核內,具有那些生物功能以及其相關之進核機轉與調控機制,仍然不甚清楚。為延續探討二者間的相關性,本論文首要目標著重在探索核內?Ⅱ-tubulin蛋白對於經由CBF-1所媒介的Notch訊息傳遞路徑所扮演的角色。 首先,為了進一步了解核內?Ⅱ-tubulin蛋白,實驗第一部份即構築帶有"標誌"以及"核內位址訊號" (nuclear localization signal; NLS)的?Ⅱ-tubulin表現質體,利用短暫轉染方式送入細胞來產生外生性核內?Ⅱ-tubulin融合蛋白。然而經由實驗結果顯示:NLS無法有效地將外生性?Ⅱ-tubulin融合蛋白帶入細胞核內,而是經由其他進核機制來達成。同時,我們也應用RNA interference原理,採Luciferase reporter assay實驗結果來證實能有效Knock down ?Ⅱ-tubulin 蛋白表現的siRNA,其會抑制經由CBF-1所媒介的Notch訊息傳遞路徑。此結果也間接說明?Ⅱ-tubulin蛋白會去提升經由CBF-1所媒介的Notch訊息傳遞路徑。 此外,為進一步探索Notch訊息路徑對於血球前驅細胞增生發展過程中的調控影響,利用本實驗室於先前建立的K562/HA-N1IC細胞株,分別採用老鼠活體實驗和流式細胞儀以及偵測細胞週期相關調控因子的蛋白表現等實驗進行研究。實驗結果顯示:在老鼠活體實驗中,注射表現N1IC的細胞所形成之腫瘤明顯小於控制組,此結果顯示Notch訊息路徑似乎在其中扮演著腫瘤抑制(tumor suppressor)角色。以PI 染色經由流式細胞儀偵測,在發現Notch訊息路徑活化情況下,K562細胞的G0/G1 phase細胞分佈情形明顯增加。而在細胞週期相關調控因子的蛋白表現方面,活化的Notch訊息路徑會抑制CDK2、CDK4、F2F1蛋白表現,並促使Rb、 c-Myc蛋白表現量增加。綜合以上實驗結果,活化的Notch訊息表現會經由Rb/E2F路徑去調控K562細胞株的細胞週期,並進而減緩其細胞增生速率。

關鍵字

Notch訊息路徑

並列摘要

Abstract Notch signaling plays a critical role in maintaining the balance between cell proliferation, differentiation, and apoptosis; hence, perturbed Notch signaling may contribute to tumorigenesis. In previous study, our laboratory constitutively overexpressed active Notch1 in K562 cells to explore the effects of Notch1 signaling on hematopoietic cell growth and to investigate the underlying molecular mechanisms. The yeast-two hybrid system was used to search the Notch1 receptor intracellular domain (N1IC)-associated protein. The nuclear ?Ⅱ-tubulin was found as a candidate of N1IC-associated proteins. The presence of ?Ⅱ-tubulin in nuclei is correlated with cancerous state of cells. However, the mechanisms that the function of ?Ⅱ-tubulin locating in the nucleus and orchestrate the multiple molecules insults required for progression of hematopoietic cells still are not clear. To investigate the relationship between Notch signaling and??Ⅱ-tubulin, the expression plasmids of ?Ⅱ-tubulin with an nuclear localization signal (NLS)sequence were constructed. This NLS tag did not increase the nuclear localization of ?Ⅱ-tubulin . The exogenous ?Ⅱ-tubulin protein expression was knocked down by the cotransfecton of ?Ⅱ-tubulin-expressing construct and RNAi expression plasmids with several target sequences of ?Ⅱ-tubulin. Furthermore, SiRNAs against ?Ⅱ-tubulin suppressed the CBF-1 mediated transactivation activity of N1IC by reporter assay. On the other hand, we showed that overexpression of Notch1 was able to inhibit the growth of hematopoietic cells in vitro and in vivo. The overexpressed active Notch1 in K562 cells decreased expression CDK2, CDK4 and the E2F1 protein. Up-regulation of retinoblastoma protein expression was observed. Furthermore, the constitutively active Notch1 could inhibit the growth of K562 cells in nude mice and cell cycle distribution analysis showed an increase in G0/G1 phase. Taken together, these results suggested that nuclear??Ⅱ-tubulin enhanced the CBF-1 mediated transactivation activity of N1IC and the classical NLS-dependent pathway does not promote the nucleus localization of ?Ⅱ-tubulin protein to modulated Notch signaling. We also demonstrated that Notch1 signaling results in growth inhibition of hematopoietic cells both in vitro and in vivo, affecting Rb/E2F pathway that control cell proliferation.

並列關鍵字

betaⅡ-tubulin ； Notch ； CBF1

參考文獻

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