Role of the Acetyltransferase p300 in Cellular Responses

性状（申请人提供）：影响微管动力学的药物是最重要的化疗药物之一。临床相关的抗微管药物包括紫杉醇（紫杉醇）、长春花生物碱（长春碱和长春新碱）、诺考达唑和秋水仙碱。这些试剂触发一个检查点，即纺锤体检查点，其监测染色体与纺锤体的附着，并在有丝分裂中诱导阻滞，通常随后是凋亡。参与这一检查点的几个细胞因子最近已被确定，然而，纺锤体组装错误或染色体附着到纺锤体参与细胞周期机制的确切分子机制仍有待阐明。我们的新发现，具有乙酰转移酶活性的转录辅激活因子p300，增强了紫杉醇引起的有丝分裂阻滞。 属于p300家族的乙酰转移酶通过调节许多细胞周期调控基因的转录，在多种信号转导途径中参与传递适应性反应。我们现在证明，p300协会与有丝分裂和间期微管，它乙酰化微管蛋白，它有利于微管蛋白聚合在紫杉醇依赖性测定。此外，p300水平及其与微管的关联在紫杉醇处理的细胞中显著增加。基于这些结果，我们假设p300作为一个重要的效应的敏感性的肿瘤细胞紫杉醇，通过其与微管蛋白和通过其作为一个转录因子的活动。为了验证这一假设，我们将：1）鉴定负责与微管相互作用的p300区域，并产生具有破坏的微管蛋白结合能力的突变体（分别丧失或获得功能）。2)研究这些突变体如何影响紫杉醇处理细胞的细胞骨架结构，纺锤体组装和乙酰化转录因子的核输入。3)定义p300参与紫杉醇诱导的细胞周期停滞和细胞凋亡的机制，并确定紫杉醇下游发生的受p300影响的分子事件。4)提供理由和战略， 设计分子，例如模拟p300对细胞凋亡作用的肽，能够增强对紫杉醇的化学敏感性。由于有丝分裂纺锤体抑制剂构成了一类不断增长的抗癌药物，因此了解耐药和敏感性的分子机制至关重要。因此，本申请中提出的研究预计具有重要的临床意义。

DESCRIPTION (provided by applicant): Drugs that affects the microtubule dynamics constitute one of the most important classes of chemotherapeutic agents. Anti-microtubule drugs of clinical relevance include paclitaxel (taxol), vinca alkaloids (vinblastin and vincrisitn), nocodazole and colchicine. These agents trigger a checkpoint, the spindle checkpoint, which monitors he attachment of chromosomes to the spindle, and elicits arrest in mitosis generally followed by apoptosis. Several cellular factors which participate in this checkpoint have recently been identified, however, the exact molecular mechanisms through which errors in spindle assembly, or chromosomes attachment to the spindle engage the cell cycle machinery remain to be elucidated. We made the novel finding that a transcription coactivator possessing acetyltransferase activity, p300, enhances the mitotic arrest elicited by taxol. Acetyltransferases belonging to the p300 family have been implicated in conveying adaptive responses in a variety of signal transduction pathways, through regulation of transcription of many cell-cycle regulatory genes. We now demonstrate that p300 associates with mitotic and interphase microtubules, it acetylates tubulin, and it favors tubulin polymerization in a taxol-dependent assay. Moreover p300 levels and its association with microtubules are significantly increased in taxol treated cells. Based on these results we hypothesize that p300 acts as an important effector of sensitivity of tumor cells to taxol, through its association with tubulin and through its activity as a transcription factor. To test this hypothesis we will: l)Identify the regions of p300 responsible for its interaction with microtubules and generate mutants with corrupted tubulin-binding ability (loss or gain of function, respectively). 2) Study how these mutants influence cytoskeleton architecture, spindle assembly and nuclear import of acetylated transcription factors in taxol treated cells. 3)Define the mechanisms by which p300 participates in cell cycle arrest and apoptosis induced by taxol and identify the molecular events occurring downstream of taxol which are influenced by p300. 4)Provide a rationale and a strategy for the design of molecules, such as peptides which mimics p300 effects on apoptosis, able to enhance chemosensitivity to taxol. Since mitotic spindle inhibitors constitute a growing class of anti-cancer agents, it is essential to understand molecular mechanisms of resistance and sensitivity. Thus, studies proposed in this application are expected to have important clinical implications.