The general objective of this research proposal is to elucidate the role of chromosomal translocations involving the c-myc gene in the pathogenesis of B-cell neoplasia. In particular, the following lines of investigation will be pursued: 1) Mechanism of c-myc oncogene activation. Chromosomal translocations are consistently associated with removal or mutation of a specific 400 bp. 5' region of the c-myc gene. The functional consequences of these mutations on c-myc gene regulation will be investigated by studying: i) whether differently 5' mutated c- myc alleles are abnormally regulated at the level of block of transcript elongation, RNA stability or translation in transfected cells; and ii) whether the 5' mutated region is involved in binding to regulatory (RNA- or DNA-binding) proteins and the binding is affected by the mutations.; 2) Mechanism of c-myc gene autoregulation and its inactivation in tumor cells. c-myc gene expression is regulated by a negative autoregulatory mechanism and this mechanism is blocked in tumor cells carrying either normal or mutated c-myc genes. We plan to further characterize this mechanism and the cause of its inactivation by; i) identifying the c-myc regulatory domains which are the targets for autoregulation of transcription; and ii) determining whether the loss of c-myc autoregulation is a dominant or recessive trait in tumor cells and whether it is controlled by a specific chromosome; 3) Identification of genes regulated by c-myc. We will attempt to identify the genes which are directly regulated by the heterodimeric myc/max transcriptional complex by isolating myc/max-binding sequences (300-400 bp.) from total genomic DNA by the """"""""genomic PCR"""""""" purification method: 4) Relationship between c-myc activation and p53 inactivation in BL. Loss/inactivation of the p53 tumor suppressor gene is specifically and consistently associated with those B cell malignancies which carry activated c-myc oncogenes and preliminary observations suggest that c-myc expression may regulate p53. Based on these observations we will: i) establish whether and by which mechanism c-myc regulates p53 gene expression by studying p53 expression in cell stably transfected with constitutive or inducible c-myc vectors; and ii) determine the effects of p53 inactivation and the combined effects of p53 inactivation and c-myc activation on the phenotype (differentiation, clonogenicity, tumorigenicity, c-myc autoregulation) of human B lymphoblastoid cells. These studies should extend our knowledge on the role of c-myc oncogene activation in neoplasia by providing direct information on the nature of both the genetic elements which regulate and are regulated by the c-myc gene.
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