Role of the Acetyltransferase p300 in Cellular Responses

乙酰转移酶 p300 在细胞反应中的作用

• 批准号: 6640927
• 负责人: MARIA L AVANTAGGIATI
• 金额: $ 13.9万
• 依托单位: GEORGE WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-08-06 至 2003-10-20
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6640927
• 关键词: MCF7 cell; acetylation; acyltransferase; antineoplastics; apoptosis; cell cycle; cytoskeleton; enzyme activity; fibroblasts; immunoprecipitation; intermolecular interaction; microinjections; microtubules; mutant; nuclear transfer; p53 gene /protein; paclitaxel; phosphorylation; posttranslational modifications; protein binding; protein sequence; site directed mutagenesis; transcription factor; tubulin; video microscopy

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.

描述(由申请人提供)：影响微管动力学的药物是最重要的化疗药物之一。具有临床意义的抗微管药物包括紫杉醇(紫杉醇)、长春花碱(长春花碱和长春新碱)、诺可达唑和秋水仙碱。这些药物触发一个检查点，即纺锤体检查点，该检查点监控染色体与纺锤体的连接，并导致有丝分裂停止，随后通常是细胞凋亡。最近，参与这一检查点的几个细胞因子已经被确定，然而，纺锤体组装中的错误或染色体附着到纺锤体中参与细胞周期机制的确切分子机制仍有待阐明。我们的新发现是，具有乙酰转移酶活性的转录共激活子p300可以增强紫杉醇引起的有丝分裂停滞。 乙酰基转移酶属于p300家族，通过调节许多细胞周期调控基因的转录，在多种信号转导通路中参与传递适应性反应。我们现在证明，p300与有丝分裂和间期微管相关，它乙酰化微管蛋白，并在紫杉醇依赖的实验中促进微管蛋白聚合。此外，在紫杉醇处理的细胞中，p300水平及其与微管的关联显着增加。基于这些结果，我们假设p300通过与微管蛋白的结合和作为转录因子的活性，发挥了肿瘤细胞对紫杉醇敏感性的重要效应。为了验证这一假设，我们将：L)确定p300负责与微管相互作用的区域，并产生微管结合能力受损的突变体(分别是功能丧失或功能获得)。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)明确p300参与紫杉醇诱导的细胞周期停滞和细胞凋亡的机制，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，以 分子的设计，如模拟p300对细胞凋亡的影响的多肽，能够增强对紫杉醇的化疗敏感性。由于有丝分裂纺锤体抑制剂构成了越来越多的抗癌药物，了解其耐药和敏感性的分子机制是至关重要的。因此，本申请中提出的研究有望具有重要的临床意义。