Reversion von terminaler Differenzierung und Zellproliferationskontrolle

终末分化和细胞增殖控制的逆转

• 批准号: 5228048
• 负责人: Professor Dr. Heinrich Leonhardt
• 金额: --
• 依托单位: Arbeitsgruppe Cell Biology and Epigenetics
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 1995
• 资助国家: 德国
• 起止时间: 1994-12-31 至 1999-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/5228048?language=en
• 关键词: Reversion; von; terminaler; Differenzierung; und

The goal of this proposal is the elucidation of regulatory mechanisms controlling cell cycle reentry in terminally differentiated muscle cells. We have etablished a cell-free replication system with primary cardiomyocytes and were able to detect in extracts from adult cardiomyocytes an activity inhibiting S-phase entry. We will now use this cell free system to identify this inhibitor by immunodepletion of specific candidates or by biochemical fractionation techniques. Recently, we have shown that proteins can be directly delivered to differentiated muscle cells by fusion to the viral VP22 factor. We will now test whether VP22-SV40 Tag can induce both, S-phase and mitosis, in myotubes and cardiomyocytes. The use of Tag mutants combined with an analysis of effects on known cell cycle factors should allow us to identify the key regulators preventing cell cycle progression in differentiated muscle cells. Our studies on the dynamics of DNA replication foci and the subnuclear localization of cyclin A and cdk2 suggested that they are involved in the activation of DNA replication system we will now directly add fluorescently labelled cdk2 and cyclin A to screen for association with replication foci. We will then add cyclinA/cdk2 mutants to map binding sites and test their role in S-phase progression.

本研究的目的是阐明控制终分化肌肉细胞细胞周期再进入的调控机制。我们已经用原代心肌细胞建立了一个无细胞复制系统，并能够在成人心肌细胞提取物中检测到抑制s期进入的活性。我们现在将使用这种无细胞系统通过特异性候选物的免疫消耗或生化分离技术来鉴定这种抑制剂。最近，我们已经证明蛋白质可以通过融合病毒VP22因子直接递送到分化的肌肉细胞。我们现在将测试VP22-SV40标签是否可以在肌管和心肌细胞中同时诱导s期和有丝分裂。利用标签突变体结合对已知细胞周期因子的影响的分析，应该使我们能够确定阻止分化肌肉细胞细胞周期进展的关键调节因子。我们对DNA复制病灶的动力学和细胞周期蛋白A和cdk2的亚核定位的研究表明，它们参与了DNA复制系统的激活。我们现在将直接添加荧光标记的cdk2和细胞周期蛋白A来筛选与复制病灶的关联。然后，我们将添加cyclinA/cdk2突变体来绘制结合位点，并测试它们在s期进展中的作用。