TOPOISOMERASES IN RETINOID INDUCED DIFFERENTIATION

拟开展的研究探讨拓扑异构酶在维甲酸调节细胞生长和分化机制中的作用。视黄酸是维生素A的代谢产物，维生素A是幼年期正常发育所必需的饮食因子，并在胚胎发生期间充当形态发生剂。这些研究使用了充分表征的HL-60人成髓细胞系。这些是未定型的造血前体细胞，其响应于视黄酸而经历G 0细胞周期停滞和髓样分化。我们的初步数据表明，视黄酸导致MEK（促分裂原活化蛋白激酶）激活，这是必要的后续细胞周期停滞和分化。其他人报道MAPK和拓扑异构酶II α共定位于染色质和免疫共沉淀。我们的初步研究表明，拓扑异构酶II α dn β，由于维甲酸进行了显着的上调和磷酸化。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异构体的功能仍不清楚。本研究验证了视黄酸激活MAPK磷酸化拓扑异构酶II的假说，即视黄酸诱导的细胞周期和分化调节需要整个位点特异性磷酸化。 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。拟议的研究集中在拓扑异构酶的作用，其在DNA复制中的作用历来被深入研究，但现在在转录控制中有一个新兴的作用。所提出的研究将（1）鉴定拓扑异构酶II α和β中的视黄酸调节的磷酸化位点，以确定当细胞被诱导分化时它们的氨基酸序列，（2）确定哪些磷酸化依赖于ERK 2 MAP激酶，（3）确定这些视黄酸诱导的位点特异性磷酸化中的哪些对诱导分化或G 0停滞特异，（4）确定α或β同种型上的特异性磷酸化结构域是否响应于视黄酸而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，是否可以通过操纵MAPK活化水平来分离视黄酸的分化和细胞周期效应。（4）中的研究将使用拓扑异构酶II β阴性HL-60细胞。这些细胞将用野生型或具有阻断的特异性磷酸化位点的突变型拓扑异构酶II β转染。这将测试β亚型是否执行除了α和β上的磷酸化位点之外的独特功能。这一提议代表了β上的位点的合作。这一提议代表了两个实验室的合作，Ram Ganapathi博士带来了在类维生素A诱导分化期间拓扑异构酶II同工酶的生物化学方面的专业知识，并作为化疗药物的靶点，Andrew Yen博士带来了维甲酸诱导骨髓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.