E2F1 inhibits MDM2 expression in a p53-dependent manner

MDM2 expression is down-regulated upon E2F1 over-expression, but the mechanism is not well defined. In the current study, we found that E2F1 inhibits MDM2 expression by suppressing its promoter activity. Although E2F1 binds to the MDM2 promoter, the inhibitory effect of E2F1 on the MDM2 promoter does not require the direct binding. We demonstrate that E2F1 inhibits MDM2 promoter activity in a p53-dependent manner. Knockdown of p53 in U2OS cells impairs the inhibitory effect of E2F1 on the MDM2 promoter. Consistent with this observation, E2F1 does not inhibit MDM2 promoter activity in p53-deficient H1299 cells, and the inhibition is restored when p53 is expressed exogenously. Both E2F1 and p53 are up-regulated after DNA damage stimulation. We show that such stimulation induces E2F1 to inhibit MDM2 promoter activity and promote p53 accumulation. Furthermore, inhibition of MDM2 by E2F1 promotes E2F1 induced apoptosis. These data suggest that E2F1 regulates the MDM2-p53 pathway by inhibiting p53 induced up-regulation of MDM2.     

Apoptosis induced by adenovirus-mediated p14ARF expression in U2OS osteosarcoma cells is associated with increased Fas expression

The INK4A/ARF locus on chromosome 9 is a tumor suppressor gene frequently mutated in human cancers. In order to study the effects of p14ARF expression in tumor cells, we constructed a recombinant adenovirus containing p14ARF cDNA (Adp14ARF). Adp14ARF infection of U2OS osteosarcoma cells which has wild type p53 and mutant p14ARF revealed high levels of p14 (ARF) expression within 24h. In addition, Adp14ARF-mediated expressing of p14 (ARF) was associated with increased levels of p53, p21, and mdm2 protein. Growth inhibition assays following Adp14ARF infection demonstrated that the growth of U2OS cells was inhibited relative to infection with control virus. Furthermore, TUNEL analysis as well as PARP cleavage assays demonstrated that Adp14ARF infection was associated with increased apoptosis in U2OS cell line and that it was associated with Adp14ARF induced overexpression of Fas and Fas-L. Addition of Fas-L neutralizing antibody NOK-1 decreased Adp14-mediated cell death, indicating that p14 (ARF) induction of the Fas pathway is associated with increased apoptosis. The finding that Adp14ARF infection did not induce Fas expression in U2OS/E6 and MCF/E6 cells suggests that wild type p53 expression may be necessary for Adp14ARF-mediated induction of Fas. The observation that overexpression of p53 by Adp53 infection in MCF-7 does not induce increased Fas protein levels nor apoptotic cell death suggests that p53 overexpression is required but not sufficient enough for apoptosis. These studies suggest there are other mechanisms other than induction of p53 in ARF-mediated apoptosis and gene therapy using Adp14ARF may be a promising treatment option for human cancers containing wild type p53 and mutant or deleted p14 expression.