The overall objective of this project is to determine the cellular and molecular mechanisms which mediate the synergistic effects of retinoic acid (RA) and interferon-g (IFN-g) and sodium phenylacetate (NaPA) on the differentiation of human neuroblastoma (nb) cells. The applicants' data suggest that these compounds affect different control points in regulation of expression of the N-myc oncogene and, together, can overcome nb cell heterogeneity and resistance to differentiation in response to single agent treatment. Studies showed that IfN-g treatment and RA act on distinct regions of the N-myc promoter to decrease N-myc transcription. The first specific aim is to characterize the promoter domains and protein interactions that mediate IfN-g induced downregulation of N-myc. Using transient transfections of nb cells with N-myc promoter-CAT constructs, the precise boundaries and critical nucleotide positions within the IfN- g response element (IRE) will be determined by observing the effects of specific mutations of IRE function. Biochemical analyses using gel retardation and footprinting will be performed to correlate function of these sequences with specific DNA-protein binding. Our studies have shown that NaPa impacts on the RA pathway at the nuclear level by upregulating RARb expression through the classical RV response element RAREb. The second specific aim is to establish the mechanism of this activity. The investigator will characterize the nuclear protein(s) that are induced by NaPa to bind RAREb, assess the effects of NaPa on expression of retinoid-X receptors, and determine other possible responsive domains mediating NaPa transactivation of RAREb. The third specific aim is to evaluate the consequence of the multi-drug downregulation of N-myc expression and induction of nb differentiation in an in vivo nude mouse model. The experiments will determine whether combination treatment protocols can increase the therapeutic efficacy compared with RA alone and prevent the outgrowth of differentiation resistant populations.By understanding the relationship between RA, IFNg and NaPA in controlling nb differentiation, these studies will provide a basic framework for the expanded clinical use of retinoids in a multidrug setting.
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