G1期细胞周期蛋白非依赖型和依赖型细胞周期调控机制的比较研究

The accurate transition from G1 phase of the cell cycle to S phase is crucial for the control of eukaryotic cell proliferation, and associated with development, regeneration and cancer. G1 cyclins play critical roles in G1/S transition. In our previous study, some types of cells (such as embryonic stem cells) are G1 cyclin-independent and still can proliferate nearly normally without any G1 cyclin. But fibroblasts are G1 cyclin-dependent. They stop growing completely after ablation of all G1 cyclins. It is very impotent to understand the difference of core cell cycle machinery among different cell types. However, the mechanisms involved still remain unclear. In this study, based on G1 cyclin-knockout model, we will rescue phenotypes caused by G1 cyclin deletion by overexpression of cyclin A, to understand G1/S transition mechanisms in G1 cyclin- independent and -dependent cells. We will also elucidate the molecular basis of G1 cyclin-independent proliferation by analyse and compare the protein profiles and phosphoproteomes in G1 cyclin-independent and -dependent cells, find out the potential proliferation regulated factors, and test their functions in human cancer cell lines. Moreover, we will generate a new model with only cyclin B driving whole cell cycle. This model will challenge our current knowledge about mammalian cell cycle. In summary, this study may uncover new mechanisms of G1 cyclin-independent proliferation and have a significant impact on our understanding of mammalian cell proliferation, including cancer cell proliferation.

真核细胞中G1期到S期的转换是细胞增殖的关键步骤，与发育、再生和肿瘤等密切相关，G1期细胞周期蛋白 (G1 cyclin) 在其中发挥重要的作用。以往我们已经证实G1 cyclin缺失后，胚胎干细胞等可较正常生长，为 “G1 cyclin非依赖型细胞”；而成纤维细胞却停止增殖分化，为 “G1 cyclin 依赖型细胞” 。本项目将在G1 cyclin knockout模型上，通过cyclin A补救G1 cyclin缺失后表型，来了解 G1 cyclin非依赖型和依赖型G1/S期转换机制和其意义；我们将比较这两类细胞的蛋白表达和磷酸化情况，在分子水平阐述 G1 cyclin非依赖型增殖机制，找出可能的新细胞增殖调控因子，研究其在肿瘤中的作用；我们还将建立只靠cyclin B来维持增殖的哺乳动物模型，挑战对细胞周期的传统认识。本项目对认识细胞周期核心机制和肿瘤细胞的异常增殖具有重要的意义。

在以往的工作中我们发现，不同类型细胞的增殖对G1期细胞周期蛋白的依赖程度不同，表明它们的细胞周期运行机制也不同，将之称为G1期细胞周期蛋白依赖型和非依赖型增殖模式。本项目在G1期细胞周期蛋白全缺失模型基础上，通过过表达cyclin A2来补救由G1期细胞周期蛋白缺失带来的增殖等方面表型，然后对补救中表型改变进行了分析，探索了G1期到S期的转换机制和G1期细胞周期蛋白在细胞增殖分化中的新功能；完成对G1期细胞周期蛋白非依赖型和依赖型细胞中蛋白表达和磷酸化的比较分析，通过对细胞内蛋白表达情况的分析，探索出一系列新的细胞增殖相关调控因子，为后续研究建立了基础；构建并分析单一cyclin（cyclin B）引导完成整个细胞周期的小鼠胚胎干细胞，不仅首次证实了一种极简但充分的、只靠cyclin B来维持的哺乳动物细胞周期驱动模式，挑战了以往对细胞周期的传统认知，这一全新的、极简化细胞周期运作系统还对揭示新的细胞增殖机制提供了全新角度。本项目的研究使我们从全新角度深入认识了细胞周期的增殖机制，对进一步全面阐明不同类型细胞周期的正常运行、肿瘤细胞的异常增殖以及胚胎发育和组织再生等生命过程的核心机制具有重要意义，也为解决肿瘤治疗中的难题如发掘新的治疗靶点、减少耐药性等提供了理论依据。