How eggs arrest at metaphase II: MPF stabilisation plus APC/C inhibition equals Cytostatic Factor

Macrophage migration inhibitory factor (MIF) is a ubiquitously expressed pro-inflammatory mediator that has also been implicated in the process of oncogenic transformation and tumor progression. We used a genetic approach to show that deletion of the MIF gene in mice has several major consequences for the proliferative and transforming properties of cells. MIF-deficient cells exhibit increased resistance to oncogenic transformation. The transformation defects associated with MIF deficiency can be overcome through concomitant inactivation of the p53 and Rb/E2F tumor suppressor pathways. We have produced compelling evidence that the effects of MIF on cell survival and tumorigenesis are mediated through overlapping pathways, wherein MIF and p53 functionally antagonize each other in the cell. However, the involvement of MIF in p53 function is secondary to p53-independent mechanisms controlling protein stability, DNA damage checkpoints, and the integrity of the genome. Given the broad spectrum of cell types that normally express MIF and its elevated levels at sites of chronic inflammation, this pathway may be generic for many early stage tumors.     

Escape from premature senescence is not sufficient for oncogenic transformation by Ras

Resistance of primary cells to transformation by oncogenic Ras has been attributed to the induction of replicative growth arrest. This irreversible 'fail-safe mechanism' resembles senescence and requires induction by Ras of p19ARF and p53 (refs 3-5). Mutation of either p19ARF or p53 alleviates Ras-induced senescence and facilitates oncogenic transformation by Ras. Here we report that, whereas Rb and p107 are each dispensable for Ras-induced replicative arrest, simultaneous ablation of both genes disrupts Ras-induced senescence and results in unrestrained proliferation. This occurs despite activation by Ras of the p19ARF /p53 pathway, identifying pRb and p107 as essential mediators of Ras-induced antiproliferative p19ARF/p53 signalling. Unexpectedly, in contrast to p19ARF or p53 deficiency, loss of Rb/p107 function does not result in oncogenic transformation by Ras, as Ras-expressing Rb-/-/p107-/- fibroblasts fail to grow anchorage-independently in vitro and are not tumorigenic in vivo. These results demonstrate that in the absence of both Rb and p107 cells are resistant to p19ARF/p53-dependent protection against Ras-induced proliferation, and uncouple escape from Ras-induced premature senescence from oncogenic transformation.     

Hepatitis B viral X protein overcomes inhibition of E2F1 activity by pRb on the human Rb gene promoter

Hepatitis B virus X (HBx) protein is known as an oncogenic transactivator, E2F1 as a positive regulator of the cell cycle, and pRb as a tumor suppressor. Here, we investigated the functional interactions of these proteins on the human Rb promoter. Interestingly, HBx transactivated the Rb promoter cooperatively with E2F1 in HepG2 cells but not in HeLa cells, in which the functions of p53 and pRb are inactive. Combinatorial cotransfection analyses in HepG2 cells showed that HBx overcame the inhibition of E2F1 activity by pRb but not that by p53. Domain analysis showed that aa 47-70 and aa 117-133 of HBx are important for this effect. These results suggest that HBx could inhibit the pRb tumor suppressor and increase E2F1 activity. Our data support the oncogenic potential of HBx, which may cause HBV-infected cells to grow continuously in the development of hepatocellular carcinoma.     

E2F1 and E2F3 activate ATM through distinct mechanisms to promote E1A-induced apoptosis

Deregulation of the Rb-E2F pathway occurs in many cancers and results in aberrant cell proliferation as well as an increased propensity to undergo apoptosis. In most cases, apoptosis in response to Rb inactivation involves the activation of p53 but the molecular details of the signaling pathway connecting Rb loss to p53 are poorly understood. Here we demonstrate that the E1A oncoprotein, which binds and inhibits Rb family members, induces the accumulation and phosphorylation of p53 through the DNA damage-responsive ATM kinase. As a result, E1A-induced apoptosis is significantly impaired in cells lacking ATM. In contrast, inactivation of ARF, which is widely believed to activate p53 in response to oncogenic stress, has no effect on p53 induction and only a modest effect on apoptosis in response to E1A. Both E2F1 and E2F3 contribute to ATM-dependent phosphorylation of p53 and apoptosis in cells expressing E1A. However, deregulated E2F3 activity is implicated in the DNA damage caused by E1A while E2F1 stimulates ATM- and NBS1-dependent p53 phosphorylation and apoptosis through a mechanism that does not involve DNA damage.     

Impaired DNA damage checkpoint response in MIF-deficient mice

Recent studies demonstrated that proinflammatory migration inhibitory factor(MIF) blocks p53-dependent apoptosis and interferes with the tumor suppressor activity of p53. To explore the mechanism underlying this MIF-p53 relationship, we studied spontaneous tumorigenesis in genetically matched p53-/- and MIF-/-p53-/- mice. We show that the loss of MIF expression aggravates the tumor-prone phenotype of p53-/- mice and predisposes them to a broader tumor spectrum, including B-cell lymphomas and carcinomas. Impaired DNA damage response is at the root of tumor predisposition of MIF-/-p53-/- mice. We provide evidence that MIF plays a role in regulating the activity of Cul1-containing SCF ubiquitin ligases. The loss of MIF expression uncouples Chk1/Chk2-responsive DNA damage checkpoints from SCF-dependent degradation of key cell-cycle regulators such as Cdc25A, E2F1 and DP1, creating conditions for the genetic instability of cells. These MIF effects depend on its association with the Jab1/CSN5 subunit of the COP9/CSN signalosome. Given that CSN plays a central role in the assembly of SCF complexes in vivo, regulation of Jab1/CSN5 by MIF is required to sustain optimal composition and function of the SCF complex.     

E2F and Ras synergize in transcriptionally activating p14ARF expression

The INK4a/ARF locus, which is frequently inactivated in human tumors, encodes two distinct tumor suppressive proteins, ARF and p16INK4a. ARF stabilizes and activates p53 by negating the effects of mdm2 on p53. Furthermore, its function is not restricted to the p53 pathway and it also inhibits cell proliferation in cells lacking p53. Expression of ARF is up-regulated in response to a number of oncogenic stimuli including E2F1. We show here that while oncogenic Ras does not significantly affect p1(4AR)F expression in normal human cells it activates p1(4AR)F in cells containing deregulated E2F. Moreover, oncogenic Ras and E2F1 synergize in activating p1(4AR)F expression. Activation of p1(4AR)F promoter by E2F1 persists in the absence of the consensus E2F-binding sites in this promoter, indicating that this activation also occurs through non- canonical binding sites. The activation by oncogenic Ras requires both E2F and Sp-1 activity, demonstrating the complex regulation of p14(ARF) in response to oncogenic stimuli.     

Loss of Matrix Adhesion Triggers Rapid Transformation-Selective Apoptosis in Fibroblasts

Cell–matrix and cell–cell adhesion are recognized physiological determinants of cell growth and survival. In epithelial and endothelial cell systems, oncogenic transformation has in several cases been shown to confer resistance to apoptosis upon depriving cells of substrate adhesion. We examined the effects of oncogenic transformation in adherent versus adhesion- deprived primary embryonic fibroblasts. Whereas untransformed early passage fibroblasts undergo cell cycle arrest, their Myc/Ras- or E1A/Ras-transformed counterparts rapidly enter apoptosis when placed into suspension. This phenomenon also occurs upon incubation with a soluble, RGD-containing integrin ligand and is blocked by a peptide antagonist to ICE family proteases or by aggregation of cells plated at high density. Loss of wild-type p53 modulates the kinetics but does not abrogate this death pathway. Transformation with activated Src rather than Ras rendered fibroblasts selectively resistant to adhesion-dependent apoptosis, an effect likely related to Src's role in integrin signaling, while simultaneously sensitizing the cells to radiation-induced apoptosis. Thus cell adhesion events regulate transformation-selective apoptosis in fibroblasts and provide potentially important targets for understanding and interfering with tumor cell viability.     

E2F1 plays a direct role in Rb stabilization and p53-independent tumor suppression

To better understand the role of E2F1 in tumor formation, we analyzed spontaneous tumorigenesis in p53-/-E2F1+/+ and p53-/-E2F1-/- mice. We show that the combined loss of p53 and E2F1 leads to an increased incidence of sarcomas and carcinomas compared to the loss of p53 alone. E2F1-deficient tumors show wide chromosomal variation, indicative of genomic instability. Consistent with this, p53-/-E2F1-/- primary fibroblasts have a reduced capacity to maintain genomic stability when exposed to S-phase inhibitors or genotoxic drugs. A major mechanism of E2F1’s contribution to genomic integrity lies in mediating stabilization and engagement of the Rb protein.     

Bcl-2 blocks p53-dependent apoptosis.

Adenovirus E1A expression recruits primary rodent cells into proliferation but fails to transform them because of the induction of programmed cell death (apoptosis). The adenovirus E1B 19,000-molecular-weight protein (19K protein), the E1B 55K protein, and the human Bcl-2 protein each cause high-frequency transformation when coexpressed with E1A by inhibiting apoptosis. Thus, transformation of primary rodent cells by E1A requires deregulation of cell growth to be coupled to suppression of apoptosis. The product of the p53 tumor suppressor gene induces apoptosis in transformed cells and is required for induction of apoptosis by E1A. The ability of Bcl-2 to suppress apoptosis induced by E1A suggested that Bcl-2 may function by inhibition of p53. Rodent cells transformed with E1A plus the p53(Val-135) temperature-sensitive mutant are transformed at the restrictive temperature and undergo rapid and complete apoptosis at the permissive temperature when p53 adopts the wild-type conformation. Human Bcl-2 expression completely prevented p53-mediated apoptosis at the permissive temperature and caused cells to remain in a predominantly growth-arrested state. Growth arrest was leaky, occurred at multiple points in the cell cycle, and was reversible. Bcl-2 did not affect the ability of p53 to localize to the nucleus, nor were the levels of the p53 protein altered. Thus, Bcl-2 diverts the activity of p53 from induction of apoptosis to induction of growth arrest, and it is thereby identified as a modifier of p53 function. The ability of Bcl-2 to bypass induction of apoptosis by p53 may contribute to its oncogenic and antiapoptotic activity.