GENETIC ANALYSIS OF NEOPLASTIC TRANSFORMATION BY MYC

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The c-myc proto-oncogene has been implicated in the genesis of a variety of human tumors, including lymphomas and leukemias; furthermore, its transforming activity in vitro has been clearly demonstrated. Despite compelling evidence for the involvement of the c-myc protein in cell proliferation, mitogenesis, and differentiation, its biochemical function and precise role in normal cellular growth and neoplastic transformation have yet to be established. Our working hypothesis is that c-myc exerts its effects through interaction with other, as yet unidentified, cellular components. We propose to define the nature of these components through the isolation and molecular characterization of non-transformed variants (revertants) from populations of myc-transformed cells. This strategy is based on our expectation that genetic alterations in at least some cellular "target" genes, induced by retroviral-mediated insertional mutagenesis, will impair or suppress the transforming capability of c-myc in vitro. Our long-term objectives involve the isolation and functional analysis of the gene(s) whose alteration (i.e. activation or inactivation) is responsible for the revertant phenotype. Our approach features two experimental schemes designed to facilitate the isolation of such revertants and the identification of the responsible altered genetic loci. First, the isolation of revertants resulting simply from loss or inactivation of the c-myc gene will be minimized by the use of a transformed rat fibroblast cell line containing two stably integrated (and functional) copies of c-myc. Second, we will generate revertants by retroviral-mediated insertional mutagenesis; integration of a provirus adjacent to or within a "target" gene will serve as a physical "tag" for subsequent gene isolation. A protocol for enrichment of rare non-transformed cells in a background containing a vast excess of transformed cells will be used to select phenotypic revertants (i.e. cells exhibiting normal flat morphology). Revertants will be selected for further characterization on the basis of several criteria, including resistance to retransformation by c-myc and ability to yield transforming virus upon superinfection with a replication-competent retrovirus. Somatic cell hybridization analyses will be performed to ascertain the nature of the genetic alterations responsible for the revertant phenotypes (i.e. dominance or recessiveness). We will isolate the cellular DNA sequences flanking the site of proviral insertion in revertant cell lines by molecular cloning, and, ultimately, use these sequences as probes to isolate the involved genes from expression libraries for structural and functional characterization. We anticipate that this approach will develop novel reagents with which to address several fundamental questions concerning the role of c-myc in neoplasia, the composition of the putative multistep pathway of carcinogenesis, and more broadly, the underlying mechanisms involved in human cancer.