Regulation of E2F/DP and c-Myc by tumor supressor ARF

Tumor suppressor ARF (p14ARF humans, p19ARF mouse), encoded by the Ink4a/ARF locus, is mutated or silenced in a number of human cancers. ARF stabilizes and activates the p53 tumor suppressor protein by inhibiting its negative regulator Mdm2, which ultimately results in cell cycle arrest or apoptosis. The physiological context in which ARF-Mdm2-p53 tumor suppressor pathway is activated is in response to oncogene activation. Recent studies provide evidence for p53-independent functions of ARF Mouse lacking p53 and ARF develop a more diverse spectrum of tumors as compared to mice lacking p53 or ARF. Moreover, expression of ARF in p53 -Mdm2-ARF- cells results in G1 arrest. These data suggest that there are additional targets, which are regulated by ARF.; Since cellular factors such as E2F/DP and c-myc are key mediators of cell cycle progression, I investigated whether the p53-Mdm2 independent cell cycle arrest by ARF was through the regulation of these factors. I found that ARF binds, induces nucleolar relocalization and proteolysis of DP1. ARF inhibits E2F regulated genes before a significant inhibition of cell cycle and independent of p53. Furthermore, ARF mutant analysis reveals a correlation between G1 arrest function of ARF and its ability to regulate DP1. These results, suggest that DP1 is a target of ARF regulation. However, the E2F1/DP1 complex is resistant to ARF-regulation. Coexpression of E2F1 and DP1 results in a retardation of the ARF-induced nucleolar localization. E2F1 is more stable in the presence of ARF when coexpressed with DP1. Coexpression of DP1 overcomes the ARF-inhibition of E2F1-activated transcription. Taken together, the results suggest that E2F1, DP1 and the E2F1/DP1 complex are differentially targeted by ARF.; c-myc is also a bona-fide target of ARF. ARF interacts with c-Myc, and induces its relocalization to the nucleolus. ARF inhibits c-Myc-activated transcription and also inhibits c-Myc-induced genes before inhibition of S phase. Moreover, ARF inhibits Myc-induced progression into S phase in cells lacking functional p53.; These results explain how ARF could arrest cells lacking p53 and Mdm2. Results also highlight, that the loss of ARF will promote tumor formation not only because of the loss of the ability to activate p53 but also because these cells will loose the capability to regulate proto-oncogenes such as E2F/DP and c-myc and hence will be more susceptible to getting transformed.