Synergistic effect of hydrogen peroxide on polyploidization during the megakaryocytic differentiation of K562 leukemia cells by PMA.
Synergistic effect of hydrogen peroxide on polyploidization during the megakaryocytic differentiation of K562 leukemia cells by PMA.
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DOI：
10.1016/j.yexcr.2013.06.002
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发表时间：
2013-08-15
影响因子：
3.7
通讯作者：
Miller, William Martin
中科院分区：
文献类型：
--
作者：
Ojima, Yoshihiro;Duncan, Mark Thompson;Nurhayati, Retno Wahyu;Taya, Masahito;Miller, William Martin
关键词：
人类骨髓细胞系 K562 已被广泛用作研究巨核细胞 (MK) 分化的模型，这可以通过暴露于佛波醇 12-肉豆蔻酸酯 13-乙酸酯 (PMA) 来实现。在本研究中，实时PCR分析表明，PMA诱导的K562细胞MK分化过程中过氧化氢酶（cat）的表达受到显着抑制。此外，PMA 增加了细胞内活性氧 (ROS) 浓度，表明 ROS 是 PMA 诱导分化的关键因素。将 PMA 分化的 K562 细胞暴露于过氧化氢 (H2O2)，以阐明 MK 分化过程中 ROS 的功能。有趣的是，第9天时，有H2O2的高倍性（DNA含量>4N）细胞的百分比为34.8±2.3%，比没有H2O2的细胞（21.5±0.8%）高70%。此外，在 PMA 诱导的 MK 分化的前 3 天添加 H2O2 对多倍体化的影响最大。为了阐明 H2O2 增强多倍化的机制，BrdU 测定清楚地表明 H2O2 抑制 4N 细胞分裂为 2N 细胞，随后 K562 细胞的多倍化增加。这些发现表明，H2O2 介导的多倍体化的增强是由于与 PMA 协同抑制胞质分裂。尽管H2O2在原代细胞的MK分化过程中没有增加倍性，但我们清楚地观察到cat表达在未成熟和成熟的原代MK细胞中均受到抑制，并且用抗氧化剂N-乙酰半胱氨酸处理有效地阻止和/或延迟了未成熟MK细胞的多倍化。总之，这些发现表明 MK 细胞在成熟的早期阶段对 ROS 水平更加敏感。
The human myelogenous cell line, K562 has been extensively used as a model for the study of megakaryocytic (MK) differentiation, which could be achieved by exposure to phorbol 12-myristate 13-acetate (PMA). In this study, real-time PCR analysis revealed that the expression of catalase (cat) was significantly repressed during MK differentiation of K562 cells induced by PMA. In addition, PMA increased the intracellular reactive oxygen species (ROS) concentration, suggesting that ROS was a key factor for PMA-induced differentiation. PMA-differentiated K562 cells were exposed to hydrogen peroxide (H2O2) to clarify the function of ROS during MK differentiation. Interestingly, the percentage of high-ploidy (DNA content >4N) cells with H2O2 was 34.8±2.3% at day 9, and was 70% larger than that without H2O2 (21.5±0.8%). Further, H2O2 addition during the first 3 days of PMA-induced MK differentiation had the greatest effect on polyploidization. In an effort to elucidate the mechanisms of enhanced polyploidization by H2O2, the BrdU assay clearly indicated that H2O2 suppressed the division of 4N cells into 2N cells, followed by the increased polyploidization of K562 cells. These findings suggest that the enhancement in polyploidization mediated by H2O2 is due to synergistic inhibition of cytokinesis with PMA. Although H2O2 did not increase ploidy during the MK differentiation of primary cells, we clearly observed that cat expression was repressed in both immature and mature primary MK cells, and that treatment with the antioxidant N-acetylcysteine effectively blocked and/or delayed the polyploidization of immature MK cells. Together, these findings suggest that MK cells are more sensitive to ROS levels during earlier stages of maturation.
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