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Overexpression of the oncoprotein MDM2 is found in a wide spectrum of human cancers and correlates with poor prognosis. Transgenic mice harboring MDM2 are predisposed to spontaneous tumor formation. However, the underlying mechanisms by which MDM2 promotes carcinogenesis are far from clear. Although the major tumorigenicity of MDM2 is attributed to its negative regulation of tumor suppressor p53, p53-independent activities have drawn increasing attention. Recently, we have generated second generation antisense oligonucleotides specifically targeting MDM2. These oligonucleotides have demonstrated antitumor activities in human cancers with and without functional p53, in vitro and in vivo, indicating the MDM2 has both p53-dependent and -independent tumorigenic effects. Accompanying MDM2 inhibition, there was E2F1downregulation. The present study was designed to elucidate the mechanisms of MDM2 affecting E2F1 expression. It was found that MDM2 prolonged the half-life of endogenous E2F1 protein and inhibited its ubiquitination in PC3 cells which have no p53. The observed binding between MDM2 and E2F1 is important to the effects of MDM2 on E2F1 ubiquitination, as MDM2 deletion constructs lacking the binding capabilities lose the ability to inhibit E2F1 ubiquitination. Further, the binding of MDM2 displaces SCFSKP2, the E3 ligase promoting ubiquitination of E2F1, from E2F1. Moreover, MDM2 and E2F1 are both localized in the nucleus, and deletion of the nuclear localization signal of MDM2 results in the loss of ability to increase E2F1 expression. To exclude the possible involvement of tumor suppressor pRb and p14Arf, both of which have activities regulating E2F1 and MDM2 functions, DU145 and MCF-7 (with p53 knockdown) cells, which lack functional pRb and p14Arf respectively, were treated with MDM2 oligonucleotides. After MDM2 knockdown, downregulation of E2F1 is still observed. Finally, H1299 cells with E2F1 deletion were employed to test the physiological consequences of stabilization effects of E2F1 by MDM2. It was observed that E2F1 at least partially contributed to the inhibition of cell proliferation induced by MDM2 knockdown. Moreover, E2F1 protected cells from death upon MDM2 inhibition. In conclusion, the present study provides evidence that stabilization of E2F1 protein by MDM2 may be another component in the p53-independent tumorigenicity of MDM2. This effect may be helpful for developing novel anticancer therapies based on MDM2. (Supported by NIH/NCI grant R01 CA80698)

[Proc Amer Assoc Cancer Res, Volume 46, 2005]