Oncoprotein MDM2 is a component of a negative feedback loop that regulates the activity and cellular level of tumor suppressor p53. The synthesis of MDM2 is regulated at the translational level in resting fibroblasts that are activated to reenter the cell cycle. In one class of soft tissue tumors, synthesis of MDM2 protein is enhanced by one to two orders of magnitude without a corresponding increase in mRNA level. In all cell types examined, there are two forms of mdm2 mRNA. The long form (L-mdm2) contains a 287-nucleotide 5' leader, with two upstream open reading frames (uORFs), derived from exon 1 and the short form (S-mdm2) has a 64-nucleotide leader arising from exon 2. L-mdm2 is inefficiently loaded with ribosomes in normal fibroblasts, HeLa cells and choriocarcinoma cells. The L-mdm2 leader confers inefficient ribosome loading on a reporter gene and the two uORFs cooperate synergistically to suppress translation. In the choriocarcinoma cell line, in which MDM2 expression is translationally derepressed, the synergistic interaction between the uORFs in the L-mdm2 leader is lost. The S-mdm2 mRNA is well loaded with ribosomes in the choriocarcinoma cells, but is poorly translated in normal fibroblasts and HeLa cells. Fusion constructs containing the S-mdm2 leader are well translated in all cell types, suggesting that the translational suppression in normal cells requires another element in part of the mRNA other than the 5' leader. This application proposes a series of experiments designed to define the mechanisms of translational control of the mdm2 gene in normal and neoplastic cells. The synergistic interaction between the two uORFs in L-mdm2 is of particular interest, first because it is lost in tumor cells that overexpress MDM2 protein and, second, because there is no mammalian model that is readily accessible for experimental study of the mechanism of regulation by multiple, interacting uORFs. By analogy to the regulation by multiple uORFs in the yeast GCN4 gene, the possible involvement of phosphorylation of eIF-alpha in regulation of MDM2 translation will be tested. The sequences in the S-mdm2 mRNA that are responsible for the differences in its translation between normal and tumor cells will also be defined.
