Molecular Mechanisms in Neoplasia

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DESCRIPTION (provided by applicant): Myc is an important oncogene widely studied in model systems and implicated in many human cancers. We have uncovered evidence for a robust genetic instability phenotype, involving DNA palindrome formation and gene copy number change, associated with constitutive Myc oncogene over expression, and resultant lymphomagenesis, in the chicken bursa of Fabricius. We propose to characterize this phenomenon more completely. To accomplish this general goal we have developed several tools for genomic analysis including a chicken cDNA microarray effective for array-based comparative genome hybridization (Array CGH) and genome-wide analysis of palindrome formation (GAPF) for analysis of lymphoma derived cell lines, and of tissues representing early and late stages of neoplastic bursal development in vivo. Specific planned experiments employ DT40, a myc-over expressing bursal lymphoma-derived cell line that demonstrates extensive DNA copy number change and palindrome formation that differs in different clonal isolates. We will:(a) determine the time scale over which these changes occur,(b) attempt to test effects of lower c-myc expression levels on stability, and (c) test the effects of deletion of several DNA repair genes including those demonstrated to be important for recombination in DT40: Myc is known to bind DNA at many loci, both in transcriptional regulatory regions and other regions of unknown function. We will map sites of Myc binding in the DT40 genome and determine if there is a relationship between Myc binding sites and sites of palindrome formation. We have observed that many sites of palindrome formation in bursal lymphomas and DT40 are expressed as RNA. We will adapt our array-based technology to determine whether RNA expressed from DNA palindromes formed during tumorigenesis has double-stranded character. Such dsRNAs are potential substrates for the formation of functional small interfering RNA which could play a role in gene silencing during tumor development. Finally, we will determine if Myc-ihduced instability occurs in mammalian systems. Palindrome formation will be assessed in Emu-c-myc transgenic mice which develop lympomas. In human P493-6 B-cells, in which Myc can be experimentally regulated, we will determine if induction of Myc results in palindrome formation at sites of Myc binding. In this fashion we hope to establish whether Myc-induced genomic instability merits wider exploration in human neoplasm.