TOPOISOMERASES IN RETINOID INDUCED DIFFERENTIATION

类维生素A诱导分化中的拓扑异构酶

• 批准号: 6350747
• 负责人: ANDREW YEN
• 金额: $ 30.87万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-02-01 至 2004-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6350747
• 关键词: DNA topoisomerases; active sites; cell differentiation; cell growth regulation; enzyme activity; enzyme inhibitors; isozymes; mitogen activated protein kinase; nutrition related tag; phosphoproteins; protein sequence; retinoate; tissue /cell culture

The proposed studies investigate the role of topoisomerases in the mechanism by which retinoic acid regulates cell growth and differentiation. Retinoic acid is a metabolite of vitamin A which is a dietary factor necessary for proper development in the juvenile and acts as a morphogen during embyrogenesis. The studies use the well characterized HL-60 human myeloblastic cell line. These are uncommitted hematopoietic precursor cells that undergo G0 cell cycle arrest and myeloid differentiation in response to retinoic acid. Our preliminary data show that retinoic acid causes MEK (Mitogen Activated Protein Kinase) activation, which is necessary for subsequent cell cycle arrest and differentiation. Other have reported that MAPK and topoisomerase IIalpha co-localize on chromatin and co- immunoprecipitation. Our preliminary studies show that both topoisomerase IIalpha dn beta, undergo a pronounced up-regulation and phosphorylation due to retinoic acid. Topoisomerase II alpha is known cell cycle regulator, but the function of the closely homologous beta isoform is still unknown. The proposed studies test the hypothesis that retinoic acid activates MAPK to phosphorylate .topoisomerase II, whole site-specific phosphorylation is needed to effect retinoic acid induced regulation of the cell cycle and differentiation. The proposed studies focus on the role of topoisomerases, whose role in DNA replication has historically been differentiation. The proposed studies focus on the role of topoisomerases, whose role in DNA replication has historically been intensely studied, but now has an emerging role in control of transcription. The proposed studies will (1) identify retinoic acid regulated phosphorylation sites in topoisomerase II alpha and beta to determine their amino acid sequence when cells are induced to differentiate, (2) determine which phosphorylations depend on ERK2 MAP kinase, (3) establish which of these retinoic acid-induced-site specific phosphorylations are specific for induced differentiation or G0 arrest, (4) determine if specific phosphorylated domains on the alpha or beta isoforms perform redundant or unique functions in response to retinoic acid. Studies in (3) will exploit previously reported HL-60 cells stably transfected with the cFMS receptor whether differentiation and cell cycle effects on retinoic acid can be segregated through manipulating the level of MAPK activation. Studies in (4) will use a topoisomerase IIbeta negative HL-60 cell. These cells will be transfected with wild type or with mutant topoisomerase IIbeta having specific phosphorylation sites blocked. This will test if the beta isoform performs unique functions apart from alpha and the phosphorylation sites on beta this depends on. This proposal represents the collaboration of sites on beta this depends on. This proposal represents the collaboration of two laboratories, that of Dr. Ram Ganapathi, who brings expertise on the biochemistry of topoisomerase II isoenzymes during retinoid induced differentiation and as targets for chemotherapeutic drugs, and Dr. Andrew Yen, who brings expertise on the molecular and cellular biology of retinoic acid-induced myeloid differentiation.

拟议的研究调查了拓扑异构酶在视黄酸调节细胞生长和分化的机制中的作用。视黄酸是维生素 A 的代谢产物，维生素 A 是幼体正常发育所必需的饮食因子，并在胚胎发生过程中充当形态发生素。这些研究使用了特征明确的 HL-60 人类成髓细胞系。这些是未定型的造血前体细胞，在视黄酸的作用下经历 G0 细胞周期停滞和骨髓分化。我们的初步数据表明，视黄酸会导致 MEK（丝裂原激活蛋白激酶）激活，这对于随后的细胞周期停滞和分化是必需的。其他人报道 MAPK 和拓扑异构酶 IIalpha 在染色质上共定位并进行免疫共沉淀。我们的初步研究表明，拓扑异构酶 IIalpha dn beta 均因视黄酸而发生显着上调和磷酸化。拓扑异构酶 II α 是已知的细胞周期调节因子，但密切同源的 β 亚型的功能仍不清楚。所提出的研究测试了视黄酸激活MAPK以磷酸化拓扑异构酶II的假设，需要全位点特异性磷酸化来影响视黄酸诱导的细胞周期和分化的调节。 拟议的研究重点关注拓扑异构酶的作用，其在 DNA 复制中的作用历来是分化。拟议的研究重点关注拓扑异构酶的作用，拓扑异构酶在 DNA 复制中的作用历来被深入研究，但现在在转录控制中发挥着新的作用。拟议的研究将 (1) 鉴定拓扑异构酶 II α 和 β 中视黄酸调节的磷酸化位点，以确定细胞被诱导分化时的氨基酸序列，(2) 确定哪些磷酸化依赖于 ERK2 MAP 激酶，(3) 确定这些视黄酸诱导的位点特异性磷酸化中哪些对诱导分化或 G0 停滞具有特异性，(4) 确定是否有特异性磷酸化 α 或 β 同工型上的结构域响应视黄酸执行冗余或独特的功能。 (3) 中的研究将利用先前报道的用 cFMS 受体稳定转染的 HL-60 细胞，以确定是否可以通过操纵 MAPK 激活水平来分离分化和细胞周期对视黄酸的影响。 (4) 中的研究将使用拓扑异构酶 IIbeta 阴性 HL-60 细胞。这些细胞将用野生型或具有封闭的特定磷酸化位点的突变拓扑异构酶IIbeta转染。这将测试 β 同工型是否具有除 α 之外的独特功能以及其所依赖的 β 上的磷酸化位点。该提案代表了其所依赖的测试版站点的协作。该提案代表了两个实验室的合作，Ram Ganapathi 博士带来了视黄酸诱导分化过程中拓扑异构酶 II 同工酶生物化学方面的专业知识，并作为化疗药物的靶点；Andrew Yen 博士带来了视黄酸诱导骨髓分化的分子和细胞生物学方面的专业知识。