TOPOISOMERASES IN RETINOID INDUCED DIFFERENTIATION

拟议的研究探讨了拓扑异构酶在维甲酸调节细胞生长和分化机制中的作用。维甲酸是维生素A的代谢物，维生素A是青少年正常发育所必需的饮食因素，在胚胎形成过程中起着形态生成物的作用。这项研究使用了具有良好特性的HL-60人成髓细胞系。这些是未确定的造血祖细胞，在维甲酸的作用下经历G0细胞周期停滞和髓系分化。我们的初步数据显示，维甲酸导致MEK(丝裂原激活蛋白激酶)激活，这是后续细胞周期停滞和分化所必需的。另有报道称，MAPK和拓扑异构酶IIα共定位于染色质和免疫共沉淀。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IIα是已知的细胞周期调节因子，但同源的β亚型的功能尚不清楚。这些研究验证了维甲酸激活MAPK磷酸化拓扑异构酶II的假说，需要整个位点特异性的磷酸化才能影响维甲酸诱导的细胞周期和分化的调节。拟议的研究集中在拓扑异构酶的作用，其在DNA复制中的作用历来是分化的。拟议的研究重点放在拓扑异构酶的作用上，它在DNA复制中的作用历来被深入研究，但现在在转录控制方面有一个新的作用。拟议中的研究将(1)确定维甲酸调节的拓扑异构酶IIα和β中的磷酸化位点，以确定它们在诱导细胞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)确定这些维甲酸诱导的位点特异性磷酸化中哪些是诱导分化或G0停滞所特有的；(4)确定在维甲酸诱导下，α或β亚型上的特定磷酸化结构域是否发挥多余或独特的功能。(3)中的研究将探索先前报道的稳定转染cFMS受体的HL-60细胞是否可以通过控制MAPK的激活水平来分离分化和细胞周期对维甲酸的影响。(4)中的研究将使用拓扑异构酶IIbeta阴性的HL-60细胞。这些细胞将被野生型或突变的拓扑异构酶IIbeta导入，这些突变的拓扑异构酶IIbeta具有特定的磷酸化位点。这将测试β异构体是否执行除α和依赖于β的磷酸化位点之外的独特功能。这项提议代表了网站在测试版上的合作，这是它所依赖的。这项建议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同工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髓系分化的分子和细胞生物学方面的专业知识。

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.