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  • 學位論文

急性原骨髓白血病細胞株中的Notch訊息傳導系統

The members of Notch signaling system in Acute Promyelocyte Leukemia (APL) cell lines

指導教授 : 曾銘仁
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摘要


原骨髓白血病細胞株 (APL) 屬於早期的骨髓系細胞且不具有分化為成熟的能力。PKC的活化分子,12-O-tetradecanoylphorbol-13-acetate (TPA) 能夠刺激APL細胞株分化成為巨嗜細胞;維生素A的衍生物,all-trans retinoic acid (ATRA) 以及極性化合物,dimethyl sulfoxide (DMSO) 都具有促進早期骨髓系細胞分化成為成熟顆粒性細胞的能力。在過去十年間,ATRA臨床上被用來治療APL病人促使其細胞分化為成熟的細胞,不過,除了初期有好的治療效果外,大多數的病人在持續治療後,會有複發的情形並且會演變為抗RA的疾病。這些刺激性藥物如何去影響細胞分化的真正機制,目前為止仍然是不清楚的。 Notch訊息傳導一般典型的觀念被認為是會維持細胞處在未分化的狀態下,而且在很多不同的組織或細胞中如同一個開關能夠控制細胞命運的決定,Notch訊息傳導途徑包含了Notch受體蛋白,Notch受質蛋白,細胞內作用分子與Notch修飾分子。在哺乳動物中,Notch基因家族包含了4個相關的基因,分別為Notch-1至-4,和Notch受質蛋白一樣能被轉譯成膜蛋白;受質-受體間相互作用所引起Notch受體蛋白的活化會促使作用蛋白-CSL (CBF1, Su(H), Lag-1) 蛋白扮演抑制分子或者是轉錄活化分子上的角色改變,並且進一步地正向調節下游的標的基因。我們有興趣於探討TPA和ATRA或DMSO刺激APL細胞株分化的過程中,Notch訊息傳導所扮演的角色。在本論文中,HL-60和KG-1細胞在2x 10-8 M TPA刺激二天後能夠分化成為巨嗜細胞,而HL-60細胞在10-6 M ATRA或1.5% DMSO刺激五天後能夠分化成為顆粒性細胞,但是卻不會影響KG-1細胞的分化。我們用細胞離心抹片機將細胞離下,並以劉氏染色法來確認APL細胞株的分化,從細胞中分離全部的RNA並利用RT-PCR的技術來闡明APL細胞株分化前後Notch分子、Notch受質和Notch修飾分子在轉錄層次上的變化。 我們發現,在APL細胞株中同時會表現Notch受體蛋白:Notch-1、-2、-4,Notch受質蛋白Jagged-1,以及Notch修飾分子manic fringe。當APL細胞分化成兩分支細胞的其中之一時,Notch受體蛋白:Notch-1、-2、-4,Notch受質蛋白Jagged-1以及Notch修飾分子manic fringe在轉錄層次上並不會受影響。我們的結果並沒有顯示出APL在不同分化時期與Notch訊息傳導在轉錄層次調節上的相關性;而近來的研究則顯示出Notch訊息傳導活性的調控是在後轉譯修飾機制上,尤其是對於蛋白質的修飾上。因此,我們推測後轉譯層次的調控Notch訊息傳導系統以及經由誘導細胞分化的誘導物所活化的訊息分子與Notch訊息傳導間相互的調控,才是主要會影響APL細胞的分化。

並列摘要


The promyelocytic cells in acute promyelocytic leukemia (APL) devoid the ability for terminal differentiation. The PKC activator, 12-O-tetradecanoylphorbol-13-acetate (TPA) can induce the differentiation of APL cell lines into macrophage. The Vitamin A derivatives, all-trans retinoic acid (ATRA), and the polar-planer compound, dimethyl sulfoxide (DMSO), have the ability to induce granulocyte maturation from promyelocytic cells. In the past decade, clinical therapy for APL patients used ATRA to induce cells maturation. Nevertheless, despite an initial good response, most patients that received continuous treatment with ATRA relapse and develop RA-resistant disease. The real mechanism of those inducer drugs influence cells differentiation still unsolved. The classical view holds that Notch signaling keeps cells in an undifferentiated state and as an important switch controlling cell fate decisions in a wide variety of tissues and cell types. Notch signaling pathway involves Notch receptors, Notch ligands, intracellular effectors and Notch modulators. In mammals the Notch gene family comprise four related genes, Notch1-4, as the Notch ligands that encode transmembrane proteins. Ligand-receptor interaction mediated Notch activation ultimately leads to the conversion of effectors-CSL (CBF1, Su(H), Lag-1) proteins, from repressors to transcriptional activators, and subsequent up-regulation of downstream targets. We are interesting in exploring the role of Notch signaling on the ATRA or DMSO- and TPA-induced differentiation of APL cell lines. In this study, HL-60 and KG-1 cells were induced to differentiate into macrophage-like cells after exposure to 2x 10-8 M TPA for 2 days. Five days of treatment with 10-6 M ATRA or 1.5 % DMSO can stimulate HL-60 to differentiate into granulocyte cells without affecting KG-1 cells. The differentiation of APL cell lines was confirmed by Liu’s stain on cytospin preparation. The total RNA from those cells was isolated and the transcriptional levels of Notch molecules, Notch ligands, as well as Notch modulates were elucidated by the RT-PCR methodology. We found that both APL cell lines expressed Notch receptors: Notch-1, -2, -4; Notch ligand Jagged-1 and Notch modulator manic fringe. After differentiation of APL cells to either lineage, the transcriptional levels of Notch receptors: Notch-1, -2, -4; Notch ligand Jagged-1 and Notch modulator manic fringe were not affected in this study. Our data may not indicate the regulation in the transcriptional levels of Notch system correlate to the differentiation stages of APL cells. The post-translational mechanisms, especially proteins modification, were implicated to be the major candidates for regulation of Notch signaling activity in recent studies. We assume that the post-translational regulation of Notch signaling system and interaction of the signaling molecules activated by differentiation inducers with molecules in Notch pathway are the players involve in inducing differentiation of APL cells.

參考文獻


Artavanis-Tsakonas, S., Matsuno, K., Fortini, M. E. (1995) Notch signaling. Science 268:225-232.
Beg, A. A., Baltimor, D. (1996) An essential role for NF-kappaB in preventing TNF-alpha-induced cell death. Science 274:782-784.
Bell, R. M., Burns, D. J. (1991) Lipid activation of protein kinase C. J. Biol. Chem. 266:4661-4664.
Bennett, J. M., Catovsky, D., Daniel, M. T., Flandrin, G., Galton, D. A., Gralnick, H. R., Sultan, C. (1985) Proposed revised criteria for the classification of acute myeloid leukemia. Ann. Intern. Med. 103:620-625.
Ceresa, B. P., Schmid, S. L. (2000) Regulation of signal transduction by endocytosis. Curr. Opin. Cell. Biol. 12:204-210.

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