Determining the mechanisms linking cell growth to the cell cycle in the liver

项目概要 在 DNA 复制之前，细胞生长会在 G1/S 转变时触发人类细胞分裂。这个过程很重要 因为它决定了增殖细胞的大小，这对其生理功能很重要。较大 细胞，包括巨噬细胞和肝细胞，通常具有与它们的基因组成比例的额外基因组拷贝。 细胞尺寸增加。通常，如此大的细胞通过触发 DNA 合成来维持其 DNA 与细胞质的比例， 但不分裂，细胞大小与其倍性成比例。然而，虽然细胞大小和倍性经常 相关的，维持 DNA 与细胞质比例的功能尚不清楚。此外，我们不知道 尽管已经确定了许多关键的细胞周期，但控制DNA与细胞质比率的分子机制 调节蛋白。在这里，我们建议确定 DNA 与细胞质比率的函数和 通过检查小鼠肝细胞，揭示体内细胞生长与 DNA 复制之间的调节机制 发育和再生环境。这项工作的科学前提是我最近的一项突破 实验室致力于了解细胞生长如何引发分裂。与增长预期相反 增加 Cyclin D-Cdk4,6 活性，我们发现细胞生长反而会稀释细胞周期抑制剂 Rb 以触发 培养细胞的分裂。我们对体外 Rb 稀释机制的发现提出了这样的问题：该机制是否 在体内运作。在这里，我们建议明确测试 Rb 稀释度，以及一个替代模型，其中小 细胞激活 p38 以抑制小鼠肝细胞的细胞分裂。我们将测量肝细胞大小的变化 以及在一系列 Rb1 已被激活的小鼠品系中，细胞生长如何与细胞周期进展相结合 有条件地删除、敲除或过度表达。初步数据表明，对于肝细胞 相同倍性时，DNA与细胞质的比例与Rb1基因剂量呈负相关，与Rb- 稀释模型。我们将使用这些改变 DNA 与细胞质比率的遗传模型来测试其功能 肝脏。更具体地说，我们将使用诱导型敲低和过度表达等位基因来生成肝细胞 比野生型更大或更小，并且 DNA 与细胞质的比例异常。然后我们将执行一个 肝功能和再生测试组。这很重要，因为异常的 DNA 与细胞质的比例 与各种病理状态相关，但其在任何细胞类型中的体内功能仍不清楚。综合起来， 拟议的工作很重要，因为确定细胞生长如何触发细胞分裂是一个基础 细胞和发育生物学问题。它的理解也将提供对癌症的深入了解，其中 流程监管不当。

PROJECT SUMMARY Cell growth triggers human cell division at the G1/S transition before DNA is replicated. This process is important because it determines the size of proliferating cells, which is important for their physiological functions. Larger cells, including macrophages and hepatocytes, often have additional copies of their genome in proportion to their increased cell size. Typically, such large cells maintain their DNA-to-cytoplasm ratio by triggering DNA synthesis, but not division, at cell sizes in proportion to their ploidy. However, while cell size and ploidy are frequently correlated, the function of maintaining the DNA-to-cytoplasm ratio is unclear. Moreover, we do not know the molecular mechanisms controlling the DNA-to-cytoplasm ratio despite having identified many key cell cycle regulatory proteins. Here, we propose to determine both the function of the DNA-to-cytoplasm ratio and the regulatory mechanisms linking cell growth to DNA replication in vivo by examining mouse hepatocytes in developmental and regenerative contexts. The scientific premise of this work is a recent breakthrough that my laboratory made in understanding how cell growth triggers division. Contrary to expectations that growth would increase Cyclin D-Cdk4,6 activity, we found instead that cell growth dilutes the cell cycle inhibitor Rb to trigger division in cultured cells. Our discovery of the Rb dilution mechanism in vitro raises the question if this mechanism operates in vivo. Here, we propose to definitively test the Rb dilution, and an alternative model in which small cells activate p38 to inhibit cell division in mouse hepatocytes. We will measure changes in hepatocyte cell size and how cell growth is coupled to cell cycle progression in a series of mouse lines in which Rb1 has been conditionally deleted, knocked down or over-expressed. Preliminary data indicate that for hepatocytes of the same ploidy, the DNA-to-cytoplasm ratio is inversely correlated with Rb1 gene dosage, consistent with the Rb- dilution model. We will use these genetic models that change the DNA-to-cytoplasm ratio to test its function in the liver. More specifically, we will use inducible knockdown and over-expression alleles to generate hepatocytes that are larger and smaller than wild type and have aberrant DNA-to-cytoplasm ratios. We will then perform a panel of liver function and regeneration tests. This is important because aberrant DNA-to-cytoplasm ratio is associated with various pathological states, but its function in vivo is still unclear in any cell type. Taken together, the proposed work is important because determining how cell growth triggers cell division is a fundamental question in cell and developmental biology. Its understanding will also provide insight into cancers, where this process is misregulated.