Leflunomide (LFM) is an inhibitor of mitochondrial enzyme dihydroorotate dehydrogenase (DHODH) that catalyzes the conversion of dihydroorotate to orotate coupled with the generation of reactive oxygen species (ROS) from mitochondria. We demonstrate here that LFM causes an unrestrained proliferation of mitochondria both in human osteosarcoma cell line 143B cells and rat liver derived RL-34 cells. Increases in the total mass of mitochondria per cell in LFM-treated cells were evidenced by the application of Green FM or 10-n-nonyl acridine orange to flow cytometry, an enhanced replication of mtDNA and electron microscopy. Externally added uridine improved the disturbance in cell cycle progression in LFM-treated cells, but failed to suppress such unrestrained mitochondrial proliferation. On the contrary, lapacol and 5-fluoroorotate, inhibitors of DHODH besides LFM, suppressed the biogenesis of mitochondria during the cell cycle progression. LFM, but not lapacol or 5-fluoroorotate, caused increases of the intracellular level of acetylated alpha-tubulin. These data suggest that the inhibition of DHODH may not be at least primarily related to the LFM-induced abnormal proliferation of mitochondria, and support our recent published observation that changes in the physicochemical properties of microtubules may be in someway concerned with the biogenesis of mitochondria.

中文翻译：

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来氟米特诱导哺乳动物细胞线粒体增殖的机制

来氟米特 (LFM) 是线粒体酶二氢乳清酸脱氢酶 (DHODH) 的抑制剂，可催化二氢乳清酸转化为乳清酸，同时从线粒体中产生活性氧 (ROS)。我们在此证明，LFM 在人骨肉瘤细胞系 143B 细胞和大鼠肝脏衍生的 RL-34 细胞中均导致线粒体不受限制的增殖。通过将绿色 FM 或 10-n-壬基吖啶橙应用于流式细胞术、增强 mtDNA 复制和电子显微镜，证明了 LFM 处理细胞中每个细胞线粒体总质量的增加。外部添加的尿苷改善了 LFM 处理细胞中细胞周期进程的紊乱，但未能抑制这种不受限制的线粒体增殖。相反，拉帕考和 5-氟乳清酸盐，除了 LFM 外，DHODH 抑制剂还抑制了细胞周期进程中线粒体的生物发生。LFM，但不是 lapacol 或 5-fluoroorotate，导致乙酰化α-微管蛋白的细胞内水平增加。这些数据表明 DHODH 的抑制可能至少与 LFM 诱导的线粒体异常增殖无关，并支持我们最近发表的观察，即微管理化特性的变化可能在某种程度上与线粒体的生物发生有关。