Novel functions of D-type and E-type cyclins in normal and in cancer cells

 描述(申请人提供)：哺乳动物细胞的增殖是由核心细胞周期机制驱动的。这一机制的关键组成部分是被称为细胞周期蛋白的蛋白质，它结合、激活并为细胞周期蛋白依赖性蛋白激酶(CDK)提供底物特异性。细胞周期蛋白-CDK复合体使细胞蛋白磷酸化，从而推动细胞增殖。在细胞周期的G1期运行的周期蛋白(所谓的“G1周期蛋白”)对癌症领域特别重要，因为许多致癌途径被证明与这些蛋白有关。哺乳动物细胞中存在两类G1期细胞周期蛋白：D型(细胞周期蛋白D_1、D_2和D_3)和E型(E_1和E_2)。D-细胞周期蛋白激活CDK4和CDK6，而E-细胞周期蛋白主要激活CDK2。细胞周期蛋白D-CDK4、D-CDK6和E-CDK2复合体可磷酸化一组重叠的蛋白质；细胞周期蛋白E相关的激酶被认为针对更广泛的底物。在过去，我们的实验室培育并鉴定了缺乏D型或E型细胞周期蛋白的小鼠。虽然这些动物表现出局限性的异常，但大多数细胞类型在缺乏D型或E型细胞周期蛋白的情况下正常或接近正常地增殖。我们将没有更深刻的表型归因于两个G1周期蛋白类的重叠功能。为了更好地了解G1周期蛋白在体内的功能，我们产生了条件五重基因敲除的小鼠胚胎干细胞。这些细胞使我们能够研究胚胎干细胞中所有五个G1周期蛋白的急性关闭的后果。在拟议的工作中，我们将使用这个实验系统来研究G1周期蛋白的分子功能。我们研究计划的一个重要部分是分析G1周期蛋白在小鼠和人类癌细胞中的功能。我们将重点关注胶质母细胞瘤，这是最具侵袭性和不可治愈的脑癌。我们将使用胶质母细胞瘤的小鼠模型和原代患者来源的胶质母细胞瘤细胞来研究G1周期蛋白在这种肿瘤类型中的分子功能。我们将测试抑制G1周期蛋白功能是否代表了这种致命癌症的治疗策略。我们将在三个具体目标中解决以下主要问题：在具体目标1中，我们将研究 胚胎干细胞中的G1期细胞周期蛋白。在特定的目标2中，我们将研究G1周期蛋白在分化过程中的分子功能。最后，在具体目标3中，我们将通过分析G1周期蛋白在胶质母细胞瘤细胞中的功能，将我们的研究扩展到MUS和人类患者来源的癌细胞。我们的工作可能会为胶质母细胞瘤的治疗提供新的治疗策略。这种肿瘤代表了最具侵袭性和不可治愈的脑癌，受影响患者的中位生存期目前不到一年。

 DESCRIPTION (provided by applicant): The proliferation of mammalian cells is driven by the core cell cycle machinery. The key components of this machinery are proteins called cyclins, which bind, activate and provide substrate specificity to the cyclin- dependent kinases (CDKs). Cyclin-CDK complexes phosphorylate cellular proteins, thereby driving cell proliferation. Cyclins operating during the G1 phase of the cell cycle (the so-called "G1 cyclins") are of particular importance to the cancer field, as many oncogenic pathways were shown to impinge on these proteins. Two classes of G1 cyclins operate in mammalian cells: D-type (cyclins D1, D2 and D3) and E-type (E1 and E2). D-cyclins activate CDK4 and CDK6, whereas E-cyclins activate mostly CDK2. Cyclin D-CDK4, D-CDK6 and E-CDK2 complexes phosphorylate an overlapping set of proteins; cyclin E-associated kinase is thought to target a broader spectrum of substrates. In the past, our laboratory generated and characterized mice lacking D-type or E-type cyclins. Although these animals displayed focused abnormalities, the majority of cell types proliferated normally or nearly normally in the absence of D-type or E-type cyclins. We ascribed the absence of more profound phenotypes to the overlapping functions of the two G1 cyclin classes. To better understand the in vivo functions of G1 cyclins, we generated conditional quintuple knockout mouse embryonic stem cells. These cells allow us to study the consequences of an acute shutdown of all five G1 cyclins in embryonic stem cells. In the proposed work, we will use this experimental system to study the molecular functions of G1 cyclins. An important part of our research plan are analyses of G1 cyclin functions in mouse and in human cancer cells. We will focus on glioblastoma, the most aggressive and incurable brain cancer. We will investigate the molecular functions of G1 cyclins in this tumor type, using a mouse model of glioblastoma as well as primary patient-derived glioblastoma cells. We will test whether inhibition of G1 cyclin function might represent a therapeutic strategy in this deadly cancer type. We will address the following major issues in our three Specific Aims: In Specific Aim 1, we will study the function of G1 cyclins in embryonic stem cells. In Specific Aim 2, we will investigate the molecular functions of G1 cyclins during differentiation. Lastly, in Specific Aim 3, we will extend our studies to mous and human patient-derived cancer cells, by analyzing the function of G1 cyclins in glioblastoma cells. Our work may suggest novel therapeutic strategies for treatment of glioblastoma. This tumor represents the most aggressive and incurable brain cancer, and the median survival time of the affected patients is currently less than one year.