Molecular Mechanisms of Ornithine Decarboxylase Regulation in Normal and Neoplastic Cells

Current research focuses on the mechanism by which ornithine decarboxylase (ODC) activity is induced during carcinogenesis by oncogenic ras. The relationship between Ras and ODC is well established, but the mechanism of ODC induction in response to Ras activation remains unclear. Blocking ODC activity reverts both the transformed phenotype of ras-transformed cells and carcinogenesis in an initiation/promotion model. The number of pathways known to be activated by Ras includes several signaling cascades that are attractive candidates for ODC regulation, including the PI 3-kinase pathway, which controls translation through regulation of eIF-4E, and Raf/MAPK, which activates the eIF-4E kinase Mnk1. ODC is translationally regulated and is induced in eIF-4E-overexpressing cells (4E-P2 cells). We have shown that downregulation of ODC activity reverts the transformed phenotype in 4E-P2 cells. Thus, it is possible that translational regulation of ODC is linked to ras activation through one of these pathways. Another candidate pathway is the JNK pathway, which is activated in ODC-overexpressing fibroblasts. The availability of partial-loss-of-function mutants of Ras makes it possible to activate each of these pathways specifically. In vitro experiments express Ras mutants which selectively activate each of these pathways in NIH-3T3 cells and IEC-6 epithelial cells, a physiologically relevant model which responds to these mutants, to examine the contribution of each pathway to ODC induction. In vivo, a transgenic mouse line overexpressing MEK, a downstream effector of Ras, in the skin was crossed with our transgenic line overexpressing an ODC dominant negative mutant driven by the Keratin 6 promoter. These mice are being used to test the hypothesis that ODC induction following the activation of ras, is a necessary step in carcinogenesis. Through this work we hope to establish a mechanism of ODC induction through Ras-activated signaling cascades, and examine the consequences of this induction using experimental models relevant to epithelial carcinogenesis. The results will increase understanding of the role of constitutively elevated ODC activity in progression from the normal to transformed phenotype, which is important since ODC has been identified as a potential target in chemoprevention. A second area of research deals with the role of increased ODC activity in the development of cardiac hypertrophy. Induction of ODC is known to occur quite rapidly in response to many agents that induce cardiac hypertrophy. The central hypothesis of the work is that this induction is not merely a marker but is a necessary step in the hypertrophic process, which eventually leads to myocardial dysfunction. To test this hypothesis, we have recently established two novel transgenic mouse lines. The first expresses ODC in the heart under the control of the a-myosin heavy chain promoter (a-MHC), resulting in life-long overexpression of ODC in both the atria and ventricles. The second expresses the protein antizyme, which inhibits ODC and causes its degradation, under control of the same promoter. We will use these models to determine whether overexpression of ODC in the heart is important to the development of cardiac hypertrophy. We will accomplish this by measuring morphological and genetic markers of cardiac hypertrophy in ODC-overexpressing hearts. We are also examining inhibition of ODC activity and of a- or b-agonist induced hypertrophy in the hearts of mice overexpressing antizyme. Finally, we have crossed our antizyme-overexpressing mice with a strain overexpressing a constitutively active form of the a1B-adrenergic receptor under control of the a-MHC promoter (MHC-Alpha1b mice). MHC-Alpha1b mice are known to develop hypertrophy, and a reduction in hypertrophy in double-transgenic mice would establish a clear role for ODC in the molecular mechanism of hypertrophy in this model.