Redox regulation of the G1 to S phase transition in the mouse embryo fibroblast cell cycle.
Redox regulation of the G1 to S phase transition in the mouse embryo fibroblast cell cycle.
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发表时间：
2003-05
期刊：
影响因子：
11.2
通讯作者：
Sarita G. Menon;E. Sarsour;D. Spitz;R. Higashikubo;M. Sturm;Hannah J. Zhang;P. Goswami
中科院分区：
文献类型：
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作者：
Sarita G. Menon;E. Sarsour;D. Spitz;R. Higashikubo;M. Sturm;Hannah J. Zhang;P. Goswami
研究了细胞内氧化/还原反应调节小鼠胚胎成纤维细胞周期G（0）-G（1）到S-期转变的假说。用巯基抗氧化剂N-乙酰-L-半胱氨酸（NAC）调节细胞内氧化还原状态，并使用BrdUrd脉冲追踪和流式细胞术分析测定细胞周期进程。用NAC处理12小时导致细胞内低分子量硫醇增加约6倍，氧化敏感探针二氢荧光素二乙酸酯的MFI降低，表明细胞内氧化还原状态向更还原的环境转变。NAC诱导的氧化还原状态的改变导致血清饥饿细胞中从G（0）-G（1）到S期的进展的选择性延迟，血清刺激这些细胞重新进入细胞周期，以及抑制异步培养物中从G（1）到S期的进展，而S期和G（2）+M过渡期没有显著改变。NAC治疗还显示细胞周期蛋白D1蛋白水平降低70%，p27蛋白水平增加3-4倍，这与视网膜母细胞瘤蛋白磷酸化降低相关。从NAC处理释放的细胞在释放后0和8小时之间显示二氢荧光素荧光和氧化型谷胱甘肽含量的瞬时增加，表明细胞内氧化还原状态向更氧化的环境转变。这些氧化还原状态的变化，其次是增加细胞周期蛋白D1，减少p27，视网膜母细胞瘤蛋白过度磷酸化和随后进入S期后8-12小时去除NAC。这些结果支持了哺乳动物细胞周期内的氧化还原循环可能在G（1）早期的代谢过程和G（1）调节蛋白的激活之间提供了一种机制联系，为细胞进入S期做准备。
The hypothesis that intracellular oxidation/reduction (redox) reactions regulate the G(0)-G(1) to S-phase transition in the mouse embryonic fibroblast cell cycle was investigated. Intracellular redox state was modulated with a thiol-antioxidant, N-acetyl-L-cysteine (NAC), and cell cycle progression was measured using BrdUrd pulse-chase and flow cytometric analysis. Treatment with NAC for 12 h resulted in an approximately 6-fold increase in intracellular low-molecular-weight thiols and a decrease in the MFI of an oxidation-sensitive probe, dihydrofluorescein diacetate, indicating a shift in the intracellular redox state toward a more reducing environment. NAC-induced alterations in redox state caused selective delays in progression from G(0)-G(1) to S phase in serum-starved cells that were serum stimulated to reenter the cell cycle as well as to inhibit progression from G(1) to S phase in asynchronous cultures with no significant alterations in S phase, and G(2)+M transits. NAC treatment also showed a 70% decrease in cyclin D1 protein levels and a 3-4-fold increase in p27 protein levels, which correlated with decreased retinoblastoma protein phosphorylation. Cells released from the NAC treatment showed a transient increase in dihydrofluorescein fluorescence and oxidized glutathione content between 0 and 8 h after release, indicating a shift in intracellular redox state to a more oxidizing environment. These changes in redox state were followed by an increase in cyclin D1, a decrease in p27, retinoblastoma protein hyperphosphorylation and subsequent entry into S phase by 8-12 h after the removal of NAC. These results support the hypothesis that a redox cycle within the mammalian cell cycle might provide a mechanistic link between the metabolic processes early in G(1) and the activation of G(1)-regulatory proteins in preparation for the entry of cells into S phase.