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

确定肝脏细胞生长与细胞周期之间的联系机制

基本信息

批准号：
10609398
负责人：
Jan M Skotheim
金额：
$ 35.01万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-04-01 至 2025-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10609398
关键词：
Affect Alleles Automobile Driving Blood capillaries Cell Culture Techniques Cell Cycle Cell Cycle Progression Cell Cycle Proteins Cell Proliferation Cell Size Cell division Cell model Cells Cellular biology Chemicals Complex Coupled Cultured Cells Cyclin D1 Cytoplasm DNA DNA biosynthesis Data Dependence Development Developmental Biology Diploidy Erythrocytes G1 Phase G1/S Transition Gene Dosage Gene Expression Gene Proteins Genetic Genetic Models Genome Growth Hepatectomy Hepatocyte Human In Vitro Laboratories Link Liver Liver Regeneration Macrophage Malignant Neoplasms Mammalian Cell Mass Spectrum Analysis Measures Modeling Molecular Mus Natural regeneration Pathologic Pathway interactions Phosphotransferases Physiological Ploidies Process Proliferating Proteins RB1 gene Replication Initiation Retinoblastoma Protein Series Signal Transduction Sorting Stress Testing Work cell growth cell type expectation experimental study genetic approach in vivo inhibitor insight knock-down liver cell proliferation liver function overexpression p38 Mitogen Activated Protein Kinase pathogen regenerative

项目摘要

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.

项目摘要细胞生长在复制DNA之前触发了G1/S转变的人类细胞分裂。这个过程很重要因为它决定了增殖细胞的大小，这对于它们的生理功能很重要。更大包括巨噬细胞和肝细胞在内的细胞通常具有与其基因组的其他副本细胞大小增加。通常，这样的大细胞通过触发DNA合成，维持其DNA与周期的比率但不能分裂，在细胞大小与其倍性成比例的细胞尺寸。但是，虽然细胞的大小和倍数经常相关的是，保持DNA与环质比的功能尚不清楚。而且，我们不知道尽管已经鉴定了许多关键细胞周期，但控制DNA与周期比的分子机制调节蛋白。在这里，我们建议确定DNA与周期比的功能和通过检查小鼠肝细胞在体内将细胞生长与DNA复制联系起来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突破实验室了解细胞生长如何触发分裂。与期望增长的期望相反增加细胞周期蛋白D-CDK4,6活性，我们发现细胞生长稀释了细胞周期抑制剂RB触发培养细胞的分裂。我们在体外发现RB稀释机制的发现提出了一个问题，即在体内操作。在这里，我们建议确定测试RB稀释和一个替代模型，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如何将细胞生长耦合到一系列RB1的小鼠系中的细胞周期进程有条件地删除，撞倒或过表达。初步数据表明，对于肝细胞同样的倍性，DNA与环质比与RB1基因剂量成反比，与RB-一致稀释模型。我们将使用这些改变DNA与周期比的遗传模型来测试其在肝脏。更具体地说，我们将使用诱导的敲低和过表达等位基因来生成肝细胞比野生型更大，更小，并且具有异常的DNA与环质比。然后我们将执行肝功能和再生测试面板。这很重要，因为异常的DNA与环质比为与各种病理状态相关，但其在体内功能在任何细胞类型中仍不清楚。在一起，提出的工作很重要，因为确定细胞生长如何触发细胞分裂是基本的细胞和发育生物学的问题。它的理解还将提供对癌症的见识过程被误导。