Reversion von terminaler Differenzierung und Zellproliferationskontrolle

该提案的目标是阐明控制终末分化肌细胞细胞周期折返的调控机制。我们已经建立了一个无细胞复制系统与原代心肌细胞，并能够检测到从成人心肌细胞提取物的活性抑制S期进入。我们现在将使用这种无细胞系统，通过特定候选物的免疫耗竭或通过生物化学分级分离技术来鉴定这种抑制剂。最近，我们已经表明，蛋白质可以直接交付到分化的肌肉细胞融合的病毒VP 22因子。我们现在将测试VP 22-SV 40标签是否可以在肌管和心肌细胞中诱导S期和有丝分裂。使用标签突变体结合对已知的细胞周期因子的影响的分析，应该使我们能够确定在分化的肌肉细胞中阻止细胞周期进展的关键调节因子。我们对DNA复制灶的动力学和cyclin A和cdk 2的亚核定位的研究表明，它们参与了DNA复制系统的激活，我们现在将直接加入荧光标记的cdk 2和cyclin A来筛选与复制灶的关联。然后，我们将添加cyclinA/cdk 2突变体来绘制结合位点，并测试它们在S期进展中的作用。

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.