Department of Cytophysiology, Faculty of Biology and Environmental Protection, University of Lodz, Pomorska 141/143, 90-236, Lodz, Poland.
Department of Molecular Biophysics, Faculty of Biology and Environmental Protection, University of Lodz, Pomorska 141/143, 90-236, Lodz, Poland.
Plant Cell Rep. 2020 Aug;39(8):1013-1028. doi: 10.1007/s00299-020-02545-9. Epub 2020 Apr 23.
Induction of biphasic interphase-mitotic cells and PCC is connected with an increased level of metabolism in root meristem cells of Allium cepa. Previous experiments using primary roots of Allium cepa exposed to low concentrations of hydroxyurea have shown that long-term DNA replication stress (DRS) disrupts essential links of the S-M checkpoint mechanism, leading meristem cells either to premature chromosome condensation (PCC) or to a specific form of chromatin condensation, establishing biphasic organization of cell nuclei with both interphase and mitotic domains (IM cells). The present study supplements and extends these observations by describing general conditions under which both abnormal types of M-phase cells may occur. The analysis of root apical meristem (RAM) cell proliferation after prolonged mild DRS indicates that a broad spectrum of inhibitors is capable of generating PCC and IM organization of cell nuclei. These included: 5-aminouracil (5-AU, a thymine antagonist), characterized by the highest efficiency in creating cells with the IM phenotype, aphidicolin (APH), an inhibitor of DNA polymerase α, 5-fluorodeoxyuridine (FUdR), an inhibitor of thymidylate synthetase, methotrexate (MTX), a folic acid analog that inhibits purine and pyrimidine synthesis, and cytosine arabinoside (Ara-C), which inhibits DNA replication by forming cleavage complexes with topoisomerase I. As evidenced using fluorescence-based click chemistry assays, continuous treatment of onion RAM cells with 5-AU is associated with an accelerated dynamics of the DNA replication machinery and significantly enhanced levels of transcription and translation. Furthermore, DRS conditions bring about an intensified production of hydrogen peroxide (HO), depletion of reduced glutathione (GSH), and some increase in DNA fragmentation, associated with only a slight increase in apoptosis-like programmed cell death events.
诱导双相间期-有丝分裂细胞和 PCC 与洋葱根尖分生区细胞代谢水平的升高有关。先前的实验使用暴露于低浓度羟基脲的洋葱根尖，表明长期 DNA 复制应激（DRS）破坏了 S-M 检查点机制的基本环节，导致分生区细胞要么过早发生染色体凝聚（PCC），要么发生特定形式的染色质凝聚，建立具有间期和有丝分裂域的双核双相组织（IM 细胞）。本研究通过描述两种异常 M 期细胞可能发生的一般条件来补充和扩展这些观察结果。对延长的轻度 DRS 后根尖分生区（RAM）细胞增殖的分析表明，广泛的抑制剂能够产生 PCC 和 IM 核结构。这些抑制剂包括：5-氨基尿嘧啶（5-AU，胸腺嘧啶拮抗剂），其在产生具有 IM 表型的细胞方面效率最高，阿霉素（APH），一种 DNA 聚合酶 α 的抑制剂，5-氟脱氧尿苷（FUdR），胸苷酸合成酶的抑制剂，甲氨蝶呤（MTX），一种抑制嘌呤和嘧啶合成的叶酸类似物，以及胞嘧啶阿拉伯糖苷（Ara-C），通过与拓扑异构酶 I 形成切割复合物来抑制 DNA 复制。如使用基于荧光的点击化学测定法所证明的，洋葱 RAM 细胞持续用 5-AU 处理与 DNA 复制机制的加速动力学以及转录和翻译水平的显著增强有关。此外，DRS 条件会导致过氧化氢（HO）的产生加剧、还原型谷胱甘肽（GSH）耗竭以及 DNA 片段化略有增加，这与仅轻微增加的细胞凋亡样程序性细胞死亡事件有关。
