Tumor-specific translocations and deregulated expression activate the oncogene c-myc in many cancers. Genetic models and target genes identify myc as an important element in growth control. The applicant identified translation initiation factor eIF4E as a critical target of c-Myc because co-transfection with a dominant inhibitor of eIF4E (4EBP1) blocks transformation by c-Myc and eIF4E itself transforms cells. To understand mechanisms coupling growth and cell division, the applicant evaluated the response of cell cycle genes to regulation by translation initiation factors. He found translational controls of CLN3 (a cyclin D1 homologue) that couple cell division to cell growth. eIF4E is a specific myc target because its promoter contains c-Myc binding sites adjacent to a novel element (CTCTTACCCCCCCTT) at -25 that is necessary for Myc-transactivation. Furthermore, levels of eIF4E regulatory factors (4ERFs) binding this element correlate with c-Myc and protein synthesis. Thus, c-myc specifically up-regulates translation factors, and translation factors specifically regulate genes controlling the cell cycle. His data suggest that regulation of eIF4E is a crucial step in myc-induced oncogenesis.
Two aims will assess the significance of eIF4E in cell transformation by c-myc:
(Aim 1) He will identify mechanisms by which expression of constitutively active, dominant inhibitors of eIF4E block transformation by c-myc.
(Aim 2) He will evaluate growth control in c-myc -/- fibroblasts to identify the molecular basis for their defect in protein synthesis.
Two aims will focus on myc-4ERF interactions:
(Aim 3) Reporter assays will assess transcriptional functions of the -25 element (LS3). Manipulations of its position, orientation and copy number in heterologous constructs will assess its potential as an activator, repressor, initiator, or as a TATA substitute. Co-transfections with c-myc expression vectors and the eIF4E promoter constructs will test for synergism between the 4ERFs and c-Myc.
(Aim 4) 4ERFs will be cloned using DNA-binding site probes and yeast one-hybrid systems. Their molecular weights and binding site suggest they will be novel factors. Full-length clones will be identified to use in expression constructs to evaluate synergism between c-myc and the 4ERFs. Biological functions of the 4ERFs in growth regulation will be analyzed and their potential regulation by c-myc tested.
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