Kapur Nidhi, Mignery Gregory A, Banach Kathrin
Department of Physiology, Stritch School of Medicine, Loyola University Chicago, 2160 South First Ave., Maywood, IL 60153, USA.
Am J Physiol Cell Physiol. 2007 Apr;292(4):C1510-8. doi: 10.1152/ajpcell.00181.2006. Epub 2006 Nov 8.
During cell cycle progression, somatic cells exhibit different patterns of intracellular Ca(2+) signals during the G(0) phase, the transition from G(1) to S, and from G(2) to M. Because pluripotent embryonic stem (ES) cells progress through cell cycle without the gap phases G(1) and G(2), we aimed to determine whether mouse ES (mES) cells still exhibit characteristic changes of intracellular Ca(2+) concentration during cell cycle progression. With confocal imaging of the Ca(2+)-sensitive dye fluo-4 AM, we identified that undifferentiated mES cells exhibit spontaneous Ca(2+) oscillations. In control cultures where 50.4% of the cells reside in the S phase of the cell cycle, oscillations appeared in 36% of the cells within a colony. Oscillations were not initiated by Ca(2+) influx but depended on inositol 1,4,5-trisphosphate (IP(3))-mediated Ca(2+) release and the refilling of intracellular stores by a store-operated Ca(2+) influx (SOC) mechanism. Using cell cycle synchronization, we determined that Ca(2+) oscillations were confined to the G(1)/S phase ( approximately 70% oscillating cells vs. G(2)/M with approximately 15% oscillating cells) of the cell cycle. ATP induced Ca(2+) oscillations, and activation of SOC could be induced in G(1)/S and G(2)/M synchronized cells. Intracellular Ca(2+) stores were not depleted, and all three IP(3) receptor isoforms were present throughout the cell cycle. Cell cycle analysis after EGTA, BAPTA-AM, 2-aminoethoxydiphenyl borate, thapsigargin, or U-73122 treatment emphasized that IP(3)-mediated Ca(2+) release is necessary for cell cycle progression through G(1)/S. Because the IP(3) receptor sensitizer thimerosal induced Ca(2+) oscillations only in G(1)/S, we propose that changes in IP(3) receptor sensitivity or basal levels of IP(3) could be the basis for the G(1)/S-confined Ca(2+) oscillations.
在细胞周期进程中，体细胞在G(0)期、从G(1)期向S期的转变以及从G(2)期向M期的转变过程中表现出不同模式的细胞内Ca(2+)信号。由于多能胚胎干细胞在没有G(1)期和G(2)期的间隙期的情况下经历细胞周期，我们旨在确定小鼠胚胎干细胞（mES细胞）在细胞周期进程中是否仍表现出细胞内Ca(2+)浓度的特征性变化。通过对Ca(2+)敏感染料fluo-4 AM进行共聚焦成像，我们发现未分化的mES细胞表现出自发性Ca(2+)振荡。在对照培养物中，50.4%的细胞处于细胞周期的S期，一个集落内36%的细胞出现了振荡。振荡不是由Ca(2+)内流引发的，而是依赖于肌醇1,4,5-三磷酸（IP(3)）介导的Ca(2+)释放以及通过储存-操纵性Ca(2+)内流（SOC）机制对细胞内储存的再填充。使用细胞周期同步化，我们确定Ca(2+)振荡局限于细胞周期的G(1)/S期（约70%的振荡细胞，而G(2)/M期约为15%的振荡细胞）。ATP诱导Ca(2+)振荡，并且在G(1)/S期和G(2)/M期同步化的细胞中可以诱导SOC的激活。细胞内Ca(2+)储存未被耗尽，并且在整个细胞周期中三种IP(3)受体亚型均存在。用乙二醇双（2-氨基乙基醚）四乙酸（EGTA）、1,2-双（2-氨基苯氧基）乙烷-N,N,N',N'-四乙酸四乙酰甲酯（BAPTA-AM）、2-氨基乙氧基二苯硼酸、毒胡萝卜素或U-73122处理后的细胞周期分析强调，IP(3)介导的Ca(2+)释放对于细胞周期通过G(1)/S期进展是必需的。由于IP(/)受体敏化剂硫柳汞仅在G(1)/S期诱导Ca(2+)振荡，我们提出IP(3)受体敏感性的变化或IP(3)的基础水平可能是G(1)/S期局限的Ca(2+)振荡的基础。
