Opposing effects of Ras on p53: transcriptional activation of mdm2 and induction of p19ARF.

Mdm2 acts as a major regulator of the tumor suppressor p53 by targeting its destruction. Here, we show that the mdm2 gene is also regulated by the Ras-driven Raf/MEK/MAP kinase pathway, in a p53-independent manner. Mdm2 induced by activated Raf degrades p53 in the absence of the Mdm2 inhibitor p19ARF. This regulatory pathway accounts for the observation that cells transformed by oncogenic Ras are more resistant to p53-dependent apoptosis following exposure to DNA damage. Activation of the Ras-induced Raf/MEK/MAP kinase may therefore play a key role in suppressing p53 during tumor development and treatment. In primary cells, Raf also activates the Mdm2 inhibitor p19ARF. Levels of p53 are therefore determined by opposing effects of Raf-induced p19ARF and Mdm2.

Loss of p14ARF in tumor cells facilitates replication of the adenovirus mutant dl1520 (ONYX-015).

The adenovirus mutant dl1520 (ONYX-015) does not express the E1B-55K protein that binds and inactivates p53. This virus replicates in tumor cells with mutant p53, but not in normal cells with functional p53. Although intra-tumoral injection of dl1520 shows promising responses in patients with solid tumors, previous in vitro studies have not established a close correlation between p53 status and dl1520 replication. Here we identify loss of p14ARF as a mechanism that allows dl1520 replication in tumor cells retaining wild-type p53. We demonstrate that the re-introduction of p14ARF into tumor cells with wild-type p53 suppresses replication of dl1520 in a p53-dependent manner. Our study supports the therapeutic use of dl1520 in tumors with lesions within the p53 pathway other than mutation of p53.

The basic helix-loop-helix transcription factors myogenin and Id2 mediate specific induction of caveolin-3 gene expression during embryonic development.

Caveolin-3 protein is the only member of the caveolin family that shows a unique muscle-specific expression pattern, and loss of its functional activity causes muscular dystrophy. Caveolin-3 mRNA levels are dramatically increased during the formation of myotubes in the C2C12 cell line. In this study, we characterized the human caveolin-3 5'-flanking region. Promoter analyses demonstrate that the proximal E box element serves as a myogenin binding site and is both necessary and sufficient to control caveolin-3 gene transcription. Transient transfection assays indicated that overexpression of myogenin activates caveolin-3 reporter gene expression, whereas Id2 overexpression inhibited caveolin-3 promoter activation by myogenin. A mutant Id2 protein lacking the HLH domain was not capable of suppressing myogenin-mediated activation. Determination of caveolin-3 transcript distribution patterns in vivo revealed that mRNA was first detectable at day 10 of gestation in the developing somites and heart. Caveolin-3 protein in myoblasts and myotubes was expressed in both the plasma membrane and vesicular structures. During skeletal myogenesis the level of Id2, an inhibitor of differentiation, decreases, allowing the induced basic helix-loop-helix transcription factor myogenin to form transcriptionally active heterodimers that bind to the caveolin-3 promoter and thereby mediate its transcription.

Growth and cell cycle abnormalities of fibroblasts from Tangier disease patients.

We have investigated the abnormal proliferation and morphology of fibroblasts from patients with Tangier disease (TD), a high density lipoprotein (HDL) deficiency syndrome that is characterized by impairment of HDL3-mediated lipid efflux and Gi-protein-mediated signaling via phosphatidylinositol-specific phospholipase C (PI-PLC) and phospholipase D (PLD). TD fibroblasts displayed a 30% to 50% reduced in vitro growth rate and a 1.6-fold increased cell surface area. The response to different mitogens was diminished, and asynchronously growing TD fibroblasts showed 4.4+/-0.3% S-phase and 19.1+/-0.5% G2/M-phase cells compared with 9.7+/-0.6% and 7.8+/-0.5%, respectively, in controls. Monensin, but not brefeldin A, induced an S- and G2/M-phase distribution in control cells similar to that found in TD fibroblasts. This effect of monensin was accompanied by an increase of ceramide levels in controls, whereas TD fibroblasts already had a 2.5-fold increased basal ceramide concentration. Incubation of control cells with C2 ceramide and threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) mimicked the effect of monensin on the cell cycle. The inhibition of neither Gi protein function by pertussis toxin nor PLD by butanol resulted in a G2/M-phase arrest. Propranolol, known to increase phosphatidic acid levels, was ineffective in reversing the G2/M-phase arrest in TD fibroblasts. In addition, cDNA sequences and mRNA expression of the participants of PI-PLC or PLD signaling, ie, G-protein subunits alphai1, alphai2, and alphai3; phosphatidylinositol transfer proteins-alpha and -beta; and ADP ribosylation factors 1 and 3 were found to be normal. Thus, growth and cell cycle abnormalities in TD fibroblasts are likely to be related to impaired Golgi function and sphingolipid signaling rather than inoperative G-protein signal transduction. Because PDMP was also found to decrease HDL3-mediated lipid efflux in control but not TD fibroblasts, similar pathways seem to be involved in the disturbances of lipid transport and growth retardation.

Molecular cloning of human caveolin 3.

We isolated a full-length cDNA encoding human caveolin 3, which is 72% and 59% homologous to human caveolin 1 and caveolin 2, respectively. Human caveolin 3 protein contains the 'caveolin signature sequence' and the 33 amino acids spanning intramembrane domain common to all caveolins. Northern blot analysis indicates that the caveolin 3 transcript is 1.6 kb in size and exclusively detectable in muscle tissue.