Lunghi P, Tabilio A, Dall'Aglio P P, Ridolo E, Carlo-Stella C, Pelicci P G, Bonati A
Department of Clinical Sciences, Section of Hemato-Oncology, University of Parma Medical School, Parma, Italy.
Leukemia. 2003 Sep;17(9):1783-93. doi: 10.1038/sj.leu.2403032.
MAP kinase/ERK kinase (MEK)-extracellular signal-regulated kinase (ERK) kinases are frequently activated in acute myelogenous leukemia (AML), and can have prosurvival function. The purpose of this study was to induce downmodulation of MEK-ERK activation in AML primary blasts in order to detect the effect on cell cycle progression and on the apoptosis of leukemic cells. We investigated 14 cases of AML with high ERK 1/2 activity and four cases with undetectable or very low activity. After 24 h incubation of the AML blasts with high ERK activity using PD98059 (New England BioLabs, Beverly, MA, USA), a selective inhibitor of MEK1 phosphorylation, at concentrations of 20 and 40 microM, we observed a strong decrease in the levels of ERK1/2 activity. A significant decrease of blast cell proliferation compared with untreated controls was found. In contrast, the proliferation of blast cells that expressed low or undetectable levels of ERK activity was not inhibited. Time-course analysis demonstrated that the downmodulation of MEK1/2, ERK1 and ERK2 dual-phosphorylation was evident even after 3 h of treatment with 20 and 40 microM. The cleavage of poly(ADP-ribose) polymerase (PARP), an early sign of apoptosis, appeared after 18 h of PD98059 treatment at concentrations of 20 and 40 microM in eight of the 14 cases. After 24 h of treatment, cleaved PARP appeared in all 14 cases. Time-course analysis of cell cycle progression and apoptosis showed that PD98059 induced a G1-phase accumulation with low or undetectable levels of apoptosis after 24 h incubation; after 48 and 72 h incubation, a significant increase of apoptosis was observed. Thus, the primary effect of ERK downmodulation was a cell cycle arrest followed by the apoptosis of a significant percentage of the leukemic blasts. The preclinical model of leukemia treatment reported in this paper makes further comment with regard to MEK1 inhibition as a useful antileukemic target, and encourages the conducting of in vivo studies and clinical investigations.
丝裂原活化蛋白激酶/细胞外信号调节激酶（MEK）-细胞外信号调节激酶（ERK）激酶在急性髓系白血病（AML）中经常被激活，并且可能具有促生存功能。本研究的目的是诱导AML原代细胞中MEK-ERK激活的下调，以检测其对细胞周期进程和白血病细胞凋亡的影响。我们研究了14例ERK 1/2活性高的AML病例和4例活性检测不到或非常低的病例。使用MEK1磷酸化的选择性抑制剂PD98059（美国马萨诸塞州贝弗利市新英格兰生物实验室），以20和40微摩尔的浓度将ERK活性高的AML原代细胞孵育24小时后，我们观察到ERK1/2活性水平大幅下降。与未处理的对照相比，发现原始细胞增殖显著减少。相反，ERK活性低或检测不到的原始细胞的增殖未受到抑制。时间进程分析表明，即使在用20和40微摩尔处理3小时后，MEK1/2、ERK1和ERK-2双磷酸化的下调也很明显。聚（ADP-核糖）聚合酶（PARP）的裂解是凋亡的早期迹象，在14例中的8例中，用20和40微摩尔浓度的PD98059处理18小时后出现。处理24小时后，所有14例中均出现裂解的PARP。细胞周期进程和凋亡的时间进程分析表明，PD98059孵育24小时后诱导G1期积累，凋亡水平低或检测不到；孵育48和72小时后，观察到凋亡显著增加。因此，ERK下调的主要作用是细胞周期停滞，随后相当比例的白血病原始细胞发生凋亡。本文报道的白血病治疗临床前模型进一步说明了MEK1抑制作为一个有用的抗白血病靶点的情况，并鼓励进行体内研究和临床研究。
