Wojciechowski Jacek, Horky Marcel, Gueorguieva Marieta, Węsierska-Gądek Józefa
Cell Cycle Regulation Group, Institute of Cancer Research, University of Vienna, Vienna, Austria.
Department of Pathological Physiology, Faculty of Medicine, Masaryk University, Brno, Czech Republic.
Int J Cancer. 2003 Sep 10;106(4):486-495. doi: 10.1002/ijc.11290.
The aim of our study was to explore the antiproliferative and pro-apoptotic action of roscovitine (ROSC) on human breast cancer MCF-7 cells. We examined the effect of ROSC on cell proliferation, cell cycle progression, nucleolar morphology, posttranslational modifications of histones as well as on induction of apoptosis. The effects of ROSC on the argyrophilic nucleolar organizer regions (AgNORs) and nucleolar RNA of MCF-7 cells were marked: ROSC treatment changed the pattern of AgNORs in a time-dependent manner. The disintegration of nucleoli manifested by increasing number of nucleolar fragments already began at 6 hr posttreatment. This was accompanied by a redistribution of the nucleolin from the nucleolus beginning after 6 hr and preceded a decrease of histone acetylation and phosphorylation. Inhibition of DNA synthesis and accumulation of G(2)/M-arrested cells starting 6 hr posttreatment coincided with a strong increase of the p53 level and with an appearance of a few cells committed to undergo apoptosis. However, all these changes preceded the main wave of apoptosis, which occurred after 24 hr ROSC treatment as assessed by determination of the frequency of Annexin binding, activation of caspases as well as of DNA fragmentation. Onset of PARP-1 cleavage detected by immunoblotting and by immunohistochemistry 6 hr or 9 hr posttreatment, respectively, preceded for a few hours the DNA fragmentation detected in situ by TUNEL assay. Reconstitution of MCF-7 cells with caspase-3 did not change the kinetics of ROSC-induced apoptosis. Our results show that disintegration of nucleoli is an early marker of ROSC-induced changes. Cell cycle arrest precedes the main wave of apoptosis.
我们研究的目的是探索罗斯考维汀（ROSC）对人乳腺癌MCF-7细胞的抗增殖和促凋亡作用。我们检测了ROSC对细胞增殖、细胞周期进程、核仁形态、组蛋白翻译后修饰以及凋亡诱导的影响。ROSC对MCF-7细胞的嗜银核仁组织区（AgNORs）和核仁RNA的影响显著：ROSC处理以时间依赖的方式改变了AgNORs的模式。核仁崩解表现为核仁碎片数量增加，在处理后6小时就已开始。这伴随着核仁素从核仁重新分布，始于6小时后，并先于组蛋白乙酰化和磷酸化的减少。DNA合成的抑制以及G(2)/M期阻滞细胞的积累从处理后6小时开始，这与p53水平的强烈升高以及一些开始发生凋亡的细胞的出现同时发生。然而，所有这些变化都先于凋亡的主要浪潮，通过检测膜联蛋白结合频率、半胱天冬酶激活以及DNA片段化评估，凋亡主要浪潮发生在ROSC处理24小时后。通过免疫印迹和免疫组织化学分别在处理后6小时或9小时检测到的PARP-1裂解，比通过TUNEL法原位检测到的DNA片段化提前了几个小时。用半胱天冬酶-3重建MCF-7细胞并没有改变ROSC诱导凋亡的动力学。我们的结果表明，核仁崩解是ROSC诱导变化的早期标志。细胞周期阻滞先于凋亡的主要浪潮。
