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.     

Cyclin D1 is required for transformation by activated Neu and is induced through an E2F-dependent signaling pathway

The neu (c-erbB-2) proto-oncogene encodes a tyrosine kinase receptor that is overexpressed in 20 to 30% of human breast tumors. Herein, cyclin D1 protein levels were increased in mammary tumors induced by overexpression of wild-type Neu or activating mutants of Neu in transgenic mice and in MCF7 cells overexpressing transforming Neu. Analyses of 12 Neu mutants in MCF7 cells indicated important roles for specific C-terminal autophosphorylation sites and the extracellular domain in cyclin D1 promoter activation. Induction of cyclin D1 by NeuT involved Ras, Rac, Rho, extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38, but not phosphatidylinositol 3-kinase. NeuT induction of the cyclin D1 promoter required the E2F and Sp1 DNA binding sites and was inhibited by dominant negative E2F-1 or DP-1. Neu-induced transformation was inhibited by a cyclin D1 antisense or dominant negative E2F-1 construct in Rat-1 cells. Growth of NeuT-transformed mammary adenocarcinoma cells in nude mice was blocked by the cyclin D1 antisense construct. These results demonstrate that E2F-1 mediates a Neu-signaling cascade to cyclin D1 and identify cyclin D1 as a critical downstream target of neu-induced transformation.     

ASK-1 (apoptosis signal-regulating kinase 1) is a direct E2F target gene

S1P (sphingosine 1-phosphate) receptor expression and the effects of S1P on migration were studied in one papillary (NPA), two follicular (ML-1, WRO) and two anaplastic (FRO, ARO) thyroid cancer cell lines, as well as in human thyroid cells in primary culture. Additionally, the effects of S1P on proliferation, adhesion and calcium signalling were addressed in ML-1 and FRO cells. All cell types expressed multiple S1P receptors. S1P evoked intracellular calcium signalling in primary cultures, ML-1 cells and FRO cells. Neither proliferation nor migration was affected in primary cultures, whereas S1P partly inhibited proliferation in ML-1 and FRO cells. Low nanomolar concentrations of S1P inhibited migration in FRO, WRO and ARO cells, but stimulated ML-1 cell migration. Consistently, S1P1 and S1P3, which mediate migratory responses, were strongly expressed in ML-1 cells, and S1P2, which inhibits migration, was the dominating receptor in the other cell lines. The migratory effect in ML-1 cells was mediated by G(i) and phosphatidylinositol 3-kinase. Both S1P and the S1P1-specific agonist SEW-2871 induced Akt phosphorylation at Ser473. However, SEW-2871 failed to stimulate migration, whereas the S1P1/S1P3 antagonist VPC 23019 inhibited S1P-induced migration. The results suggest that aberrant S1P receptor expression may enhance thyroid cancer cell migration and thus contribute to the metastatic behaviour of some thyroid tumours.     

pRb inactivation in senescent cells leads to an E2F-dependent apoptosis requiring p73

Senescent cells in which pRb is inactivated undergo apoptosis on attempted reinitiation of DNA synthesis. To further explore the cell death resulting from loss of pRb function in senescent cells, we employed a temperature-sensitive pRb mutant protein (tspRb). We found that tspRb inactivation results in rapid E2F reactivation and subsequent S-phase reentry associated with the up-regulation of E2F target gene expression and cyclin E-dependent kinase activity. Total inhibition of cyclin-dependent kinase 2 activity results in a cell cycle arrest on pRb loss and a nearly complete suppression of apoptosis. Furthermore, blocking of E2F activity with a dominant-negative DP1 inhibits S-phase reentry and cell death following tspRb inactivation. Finally, inhibition of p73 activity abolishes apoptosis but not S-phase entry on pRb inactivation, suggesting that activation of E2F in senescent cells can result in the use of p73 as a cell death effector. Interestingly, senescent cells rescued from apoptosis maintain their altered shape and express senescence-associated beta-galactosidase despite loss of pRb function. Thus, maintenance of the terminal cell cycle arrest of senescent cells requires continuous pRb-mediated inactivation of E2F activity, the reappearance of which in these irrevocably altered cells triggers a cell death program instead of an inappropriate resumption of cell cycling.