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.
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