Wang Gang, Miskimins Robin, Miskimins W Keith
University of South Dakota School of Medicine, Division of Basic Biomedical Sciences, Vermillion, SD 57069, USA.
Biometals. 2004 Feb;17(1):15-24. doi: 10.1023/a:1024417309370.
Enhanced intracellular iron levels are essential for proliferation of mammalian cells. If cells have entered S phase when iron is limiting, an adequate supply of deoxynucleotides cannot be maintained and the cells arrest with incompletely replicated DNA. In contrast, proliferating cells that are not in S phase, but have low iron pools, arrest in late G1. In this report the mechanism of iron-dependent G1 arrest in normal fibroblasts was investigated. Cells were synchronized in G0 by contact inhibition and serum deprivation. Addition of serum caused the cells to re-enter the cell cycle and enter S phase. However, if the cells were also treated with the iron chelator deferoxamine, S phase entry was blocked. This corresponded to elevated levels of the cyclin dependent kinase inhibitor p27(Kip1) and inhibition of CDK2 activity. Expression of other cell cycle regulatory proteins was not affected, including the induction of cyclins D1 and E. When the quiescent serum starved cells were supplemented with a readily usable form of iron in the absence of serum or any other growth factors, a significant population of the cells entered S phase. This was associated with downregulation of p27(Kip1) and increased CDK2 activity. Using an IPTG-responsive construct to artificially raise p27(Kip1) levels blocked the ability of iron supplementation to promote S phase entry. Thus it appears that p27(Kip1) is a mediator of G1 arrest in iron depleted Swiss 3T3 fibroblasts. We propose that this is part of an iron-sensitive checkpoint that functions to ensure that cells have sufficient iron pools to support DNA synthesis prior to entry into S phase.
细胞内铁水平的升高对哺乳动物细胞的增殖至关重要。如果细胞在铁供应受限的情况下进入S期，就无法维持足够的脱氧核苷酸供应，细胞会因DNA复制不完全而停滞。相反，未处于S期但铁储备较低的增殖细胞会在G1晚期停滞。在本报告中，研究了正常成纤维细胞中铁依赖性G1期停滞的机制。通过接触抑制和血清剥夺使细胞同步于G0期。添加血清会使细胞重新进入细胞周期并进入S期。然而，如果细胞同时用铁螯合剂去铁胺处理，S期进入会被阻断。这与细胞周期蛋白依赖性激酶抑制剂p27（Kip1）水平升高和CDK2活性受到抑制相对应。其他细胞周期调节蛋白的表达不受影响，包括细胞周期蛋白D1和E的诱导。当在无血清或任何其他生长因子的情况下，向静止的血清饥饿细胞补充易于利用的铁形式时，大量细胞进入S期。这与p27（Kip1）的下调和CDK2活性增加有关。使用IPTG响应构建体人为提高p27（Kip1）水平会阻断铁补充促进S期进入的能力。因此，p27（Kip1）似乎是铁缺乏的瑞士3T3成纤维细胞中G1期停滞的介质。我们认为这是铁敏感检查点的一部分，其功能是确保细胞在进入S期之前有足够的铁储备来支持DNA合成。
