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

项目摘要 细胞生长在DNA复制之前的G1/S转换触发人类细胞分裂。这个过程很重要 因为它决定了增殖细胞的大小，这对它们的生理功能很重要。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它们的基因组的额外拷贝。 细胞大小增加。通常，这样的大细胞通过触发DNA合成来维持其DNA与细胞质的比例， 而不是分裂，细胞大小与其倍性成比例。然而，虽然细胞大小和倍性经常被 相关，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细胞周期，但控制DNA与细胞质比例的分子机制 调节蛋白在这里，我们建议确定DNA与细胞质比率的功能和细胞质的功能。 通过检查小鼠肝细胞中细胞生长与体内DNA复制的调节机制， 发展和再生背景。这项工作的科学前提是最近的一项突破， 在理解细胞生长如何触发分裂方面取得了巨大的成就。与预期相反， 增加细胞周期蛋白D-Cdk 4，6活性，我们发现细胞生长反而稀释了细胞周期抑制剂Rb， 在培养的细胞中分裂。我们在体外Rb稀释机制的发现提出了一个问题，如果这种机制 在体内运作。在这里，我们建议明确测试Rb稀释，以及一个替代模型，其中小 细胞激活p38以抑制小鼠肝细胞中的细胞分裂。我们将测量肝细胞大小的变化 以及在一系列小鼠品系中细胞生长如何与细胞周期进程相关联，在这些小鼠品系中，Rb 1已经被 条件性删除、敲除或过度表达。初步数据表明， 相同倍性，DNA-细胞质比与Rb 1基因剂量呈负相关，与Rb- 稀释模型我们将使用这些改变DNA与细胞质比例的遗传模型来测试其在体内的功能 肝脏更具体地说，我们将使用可诱导的敲低和过表达等位基因来产生肝细胞 它们比野生型大或小，并且具有异常的DNA与细胞质比率。然后我们将执行一个 肝功能和再生测试这很重要，因为异常的DNA与细胞质的比例是 与多种病理状态相关，但其在任何细胞类型中的体内功能仍不清楚。综合起来看， 这项工作很重要，因为确定细胞生长如何触发细胞分裂是一个基本的 细胞和发育生物学的问题。它的理解也将提供对癌症的深入了解， 过程是错误的。

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.