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
中文摘要
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。
英文摘要
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.
