The broad objectives of this proposal are to identity the DNA control elements responsible for regulating promoter activity of the N-myc oncogene in human neuroblastoma, and to determine whether derangements in this regulation have clinical significance. Preliminary studies have identified regions of the N-myc promoter that mediate its down regulation by retinoic acid (RA response clement, or RARE), and its silencing in non- expressing, nonneuroblastoma cells. In addition these results have suggested that in some cases RARE mutation may be responsible for resistance to RA induced differentiation, and that differences from the silencer binding regulatory proteins present in non-expressing cells may result in the constitutive basal N-myc expression in neuroblastoma. The first specific aim is to identify and characterize the RARE responsible for mediating the disease in N-myc transcription observed upon RA induced differentiation of neuroblastoma cells. This region has been mapped by functional genetic analysis using transient transfections of neuroblastoma cell lines before and alter RA treatment. Biochemical analyses using gel retardation and footprinting will be performed to correlate function of these sequences with specific DNA-protein binding. The critical nucleotide positions within the RARE will then be determined by observing the effect of specific mutations on RARE function and protein binding. The second specific aim is to identity the DNA sequence and protein(s) mediating the silencing of the N-myc promoter in non-neuroblastoma cells. Using N-myc expressing neuroblastoma, and non-expressing, nonneuroblastoma cell lines, genetic and biochemical analyses employed in the study of the RARE will be applied to the cell type specific silencer to determine its critical sequence. Regulatory protein(s) binding to this sequence will be characterized, and cloned by weaning of a cDNA expression library with the binding sequence probe. The third specific aim is to determine whether defects in these regulatory regions and/or DNA binding protein(s) are associated with resistance to RA induced differentiation, and correlate with adverse clinical outcome. This will be examined by PCR amplifying and sequencing RARE and cell type specific silencer elements in a panel of N- myc expressing, RA sensitive and resistant neuroblastoma cell lines, in non-expressing, non-neuroblastoma cell lines, as well as in stage IV N-myc expressing tumors (persistently aggressive with poor patient outcome), N- myc expressing stage IVS tumors (initially metastatic, but undergo N-myc down regulation and differentiation to benign glioneuroma), non- expressing, differentiated ganglioneuromas, and normal tissue. As an initial indication of silencer binding protein production, reverse transcription PCR will be used to quantitate its mRNA expression in N-myc expressing and non-expressing cell lines, as well as in tumors and normal tissue. Such knowledge should aid in patient prognostication, provide insight into the use of differentiating agents in the treatment of neuroblastoma, and suggest new therapeutic approaches based on altering tumor cell behavior by modulating oncogene expression.
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