Our recent results on the function of alternative translational forms of c-Myc have led us to change our ideas on how c-Myc functions at the molecular level to control proliferation and apoptosis. Previously, we identified and characterized a non-AUG-initiated, larger translational form of c-Myc, termed c-Myc 1, and have recently identified a downstream-initiated short form of c-Myc, lacking the N-terminal 100 amino acids, termed c-MycS. Progress during the last funding period has revealed that both of these forms appear to have both overlapping and distinct molecular and biological functions compared to the AUG-initiated c-Myc2 protein. c-MycS is especially interesting since it cannot transactivate through the c-Myc """"""""E box"""""""" DNA binding site (EMS), but does retain the ability of the full-length c-Myc proteins to stimulate proliferation and anchorage-independent growth, induce apoptosis, and rescue the slow-growth phenotype of c-myc null fibroblasts. Thus, transactivation through the EMS element is not necessary for at least some of the biological functions of c-Myc. Therefore, our hypothesis is that the alternative translational forms of c-Myc have both shared and distinct biological functions mediated by differential regulation of target genes. These differences can be exploited to dissect the critical molecular pathways responsible for the various c-Myc biological functions. The following specific aims are designed to test this hypothesis: 1) The in vitro abilities of the different translational forms of c-Myc to induce DNA synthesis, stimulate growth, immortalize primary cells, inhibit differentiation and induce genomic instability will be examined. In addition, further analysis of the effects of the different forms on apoptosis and proliferation will be examined on a per cell basis using Myc-GFP chimeras and microinjection; 2) To determine the in vivo functional significance of the alternative translational forms, mice which only express one form of c-Myc protein will be generated. In addition, the ability of c-MycS to cause tumorigenesis in mice will be examined in transgenic mice; and 3) Using differential display and microarray analysis, genes which are regulated by each of the different forms of c-Myc will be identified. Additional known target genes will also be examined. Further analysis of the non-canonical DNA binding of the c-Myc proteins will be examined. Our preliminary results and the results of experiments proposed in these specific aims will have major implications concerning the function of c-Myc. Our findings have already impacted the controversial model on the molecular function of c-Myc and will likely continue to impact the course of c-Myc studies on how c-Myc functions at the molecular level to control proliferation or cause cancer.
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