TOPOISOMERASES IN RETINOID INDUCED DIFFERENTIATION

提出的研究探讨拓扑异构酶在维甲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的代谢物，维生素a是幼鱼正常发育所必需的饮食因子，在胚胎发生过程中起形态形成的作用。该研究使用了具有良好特征的HL-60人成髓细胞系。这些是未确定的造血前体细胞，在维甲酸的作用下经历G0细胞周期阻滞和髓细胞分化。我们的初步数据表明，维甲酸引起有丝分裂原活化蛋白激酶（MEK）的激活，这是随后的细胞周期阻滞和分化所必需的。其他研究报道了MAPK和拓扑异构酶i - α在染色质上共定位和共免疫沉淀。我们的初步研究表明，由于维甲酸的作用，这两种拓扑异构酶都经历了明显的上调和磷酸化。拓扑异构酶II α是已知的细胞周期调节因子，但其密切同源的β亚型的功能尚不清楚。提出的研究验证了维甲酸激活MAPK磷酸化的假设。拓扑异构酶II，整个位点特异性磷酸化需要影响维甲酸诱导的细胞周期和分化的调节。提出的研究重点是拓扑异构酶的作用，其在DNA复制中的作用历来是分化。提出的研究重点是拓扑异构酶的作用，其在DNA复制中的作用历来被广泛研究，但现在在控制转录方面有一个新兴的作用。拟建的研究将(1)确定维甲酸调节的拓扑异构酶II α和β的磷酸化位点，以确定细胞诱导分化时它们的氨基酸序列，(2)确定哪些磷酸化依赖于ERK2 MAP激酶，(3)确定这些维甲酸诱导的位点特异性磷酸化中哪些是诱导分化或G0阻滞的特异性磷酸化。(4)确定α或β同工异构体上的特定磷酸化结构域在响应维甲酸时是否具有冗余或独特的功能。(3)中的研究将利用先前报道的稳定转染cFMS受体的HL-60细胞，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i β阴性HL-60细胞。这些细胞将被野生型或突变型拓扑异构酶ⅱβ转染，其特异性磷酸化位点被阻断。这将测试β同工异构体在α和磷酸化位点之外是否具有独特的功能。该提案代表了这所依赖的beta版网站的协作。该提案代表了两个实验室的合作，一个是Ram Ganapathi博士，他带来了维甲酸诱导分化过程中拓扑异构酶II同位酶的生物化学专业知识，并作为化疗药物的靶标，另一个是Andrew Yen博士，他带来了维甲酸诱导骨髓分化的分子和细胞生物学专业知识。

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.