Lagneaux Laurence, de Meulenaer Eric Cordemans, Delforge Alain, Dejeneffe Marielle, Massy Martine, Moerman Carine, Hannecart Baudouin, Canivet Yves, Lepeltier Marie Françoise, Bron Dominique
Laboratoire d'Hématologie Expérimentale, Institut Jules Bordet, Université Libre de Bruxelles, Brussels, Belgium.
Exp Hematol. 2002 Nov;30(11):1293-301. doi: 10.1016/s0301-472x(02)00920-7.
We evaluated the cytotoxic effect of ultrasonic irradiation at low energy on the viability of normal and leukemic cells and the potential mechanisms of action inducing this cytotoxicity.
Human leukemia cell lines (K562, HL-60, KG1a, and Nalm-6), primary leukemic cells, and normal mononuclear cells are treated by ultrasound at a frequency of 1.8 MHz during various exposure times (acoustical power of 7 mW/mL) and immediately tested for cell viability by the trypan blue exclusion assay. Apoptosis is evaluated by cell morphology, phosphatidylserine exposure, and DNA fragmentation. The mitochondrial potential, glutathione content, caspase-3 activation, PARP cleavage, and bcl-2/bax ratio are tested by flow cytometry. Cloning efficiency is evaluated by assays in methylcellulose.
The technique we describe here, using minute amounts of energy and in the absence of any chemical synergy, specifically triggers apoptosis in leukemic cells while necrosis is significantly reduced. Ultrasonic treatment of 20 seconds' duration induces a series of successive phases showing the characteristic features of apoptosis: mitochondrial transmembrane potential disturbances, loss of phosphatidylserine asymmetry, morphological changes, and, finally, DNA fragmentation. In contrast to K562 cells, for which a significant reduction of cloning efficiency is observed, the growth of hematopoietic progenitors is totally unaffected. Ultrasound treatment is also associated with depletion of cellular glutathione content, suggesting a link with the oxidative stress. Moreover, the fact that active oxygen scavengers reduce ultrasonic-induced apoptosis suggests a sonochemical mechanism.
The cell damage observed after exposure of leukemic cells to ultrasound is associated with the apoptotic process and may be a promising tool for a smooth, specific, and effective ex vivo purging of leukemic cells.
我们评估了低能量超声照射对正常细胞和白血病细胞活力的细胞毒性作用以及诱导这种细胞毒性的潜在作用机制。
人白血病细胞系（K562、HL - 60、KG1a和Nalm - 6）、原代白血病细胞和正常单核细胞在不同暴露时间（声功率7 mW/mL）下接受1.8 MHz频率的超声处理，然后立即通过台盼蓝排斥试验检测细胞活力。通过细胞形态、磷脂酰丝氨酸暴露和DNA片段化评估细胞凋亡。通过流式细胞术检测线粒体电位、谷胱甘肽含量、半胱天冬酶 - 3激活、聚（ADP - 核糖）聚合酶（PARP）裂解以及bcl - 2/bax比值。通过甲基纤维素中的检测评估克隆效率。
我们在此描述的技术，使用微量能量且不存在任何化学协同作用，特异性地触发白血病细胞凋亡，同时坏死显著减少。持续20秒的超声处理诱导出一系列连续阶段，呈现出凋亡的特征：线粒体跨膜电位紊乱、磷脂酰丝氨酸不对称性丧失、形态变化，最终是DNA片段化。与K562细胞不同，在K562细胞中观察到克隆效率显著降低，而造血祖细胞的生长完全不受影响。超声处理还与细胞内谷胱甘肽含量的消耗有关，表明与氧化应激存在联系。此外，活性氧清除剂减少超声诱导的凋亡这一事实提示了一种声化学机制。
白血病细胞暴露于超声后观察到的细胞损伤与凋亡过程相关，可能是一种用于体外温和、特异性且有效地清除白血病细胞的有前景的工具。
