Overexpression of E2F-1 inhibits progression of gastric cancer in vitro

E2F-1 plays a critical role in cell cycle regulation and other biological processes in cells. E2F-1 mediates apoptosis and suppresses tumorigenesis in many tissue types, but there are few data available on E2F-1 expression and its relationship to tumor kinetics in gastric cancer. To gain better insight into the involvement of E2F-1 in the biological characteristics of gastric tumors, we investigated the effect of E2F-1 overexpression on the progression of gastric carcinoma cells. A gastric cancer cell line stably overexpressing E2F-1 (MGC-803/E2F-1) was established. The influence of E2F-1 overexpression on in vitro cell growth was assessed by measuring cell survival, colony formation, and cell cycle progression. The results clearly show that overexpression of E2F-1 significantly inhibits cell growth and proliferation, blocking entry into the S-phase of the cell cycle. MGC-803/E2F-1 cells also had a higher apoptotic rate than control cells. In addition, E2F-1 reduced the motility and invasion of gastric cancer cells.     

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.     

Topoisomerase IIα mediates E2F-1-induced chemosensitivity and is a target for p53-mediated transcriptional repression

Mutations of the retinoblastoma tumor suppressor, pRb, or its cyclin-cyclin-dependent kinase (CDK) regulatory kinases or CDK inhibitors, allows unrestrained E2F activity, leading to unregulated cell cycle progression. However, overexpression of E2F-1 also sensitizes cells to apoptosis, suggesting that targeting this pathway may be of therapeutic benefit. Enforced expression of E2F-1 in interleukin-3-dependent myeloid cells led to preferential sensitivity to the topoisomerase II inhibitor, etoposide, which was independent of p53 accumulation. Pretreatment of the E2F-1-expressing cells with ICRF-193, a second topoisomerase II inhibitor that does not cause DNA damage, protected these cells against etoposide-induced apoptosis. However, ICRF-193 cooperated with other DNA-damaging agents to induce apoptosis. Enforced expression of E2F-1 led to accumulation of p53 protein. An E2F-1 mutant that is defective in inducing cell cycle progression also induced p53, suggesting that p53 was responding directly to E2F, and not to secondary events caused by inappropriate cell cycle progression (i.e., DNA damage). Thus, topoisomerase II inhibition and DNA damage cooperate to selectively induce apoptosis in cells that have mutations in the pRb pathway.     

Regulation in S Phase by E2F

The DNA synthetic S phase of the unperturbed cell cycle is a closed system, as compared to regulation of G1 by external growth factors. The E2F family provides internal control in S phase by transcribing genes required for deoxynucleotide triphosphate (dNTP) and DNA synthesis. Furthermore, over expression of E2F-1 activates programmed cell death (apoptosis), a safeguarding signal of aberrant growth of cells that have become carcinogenic. Mechanisms for control of E2F-1 are thus essential. The hypothesis is proposed that deoxythymidine triphosphate (dTTP) allosterically feedback controls E2F-1 to regulate both DNA synthesis and apoptosis. This may act either upon production of E2F-1 or its degradation.     

E2F1 and RNA binding protein QKI comprise a negative feedback in the cell cycle regulation

pRb/E2F1 activity is coordinately regulated during the cell cycle progression, while the molecular strategies safeguarding this pathway are not fully understood. We have previously shown that RNA binding protein QKI inhibits the cell proliferation and promotes the differentiation of gastrointestinal epithelium, suggesting a role of QKI in cell cycle regulation. Here we found that with the cell entry into S phase, QKI expression increased both at the mRNA and protein levels, which was reminiscent of cyclin E expression. Forced expression of E2F1 increased the endogenous level of QKI. Promoter luciferase assay and ChIP analysis identified that the -542~-538 E2F1 binding site was responsible for the upregulation. Increased QKI expression by E2F1, in turn, reduced the E2F1 activity and delayed S-phase entry, forming a negative feedback. As a gene expression regulator, QKI overexpression increased p27, while it decreased cyclin D1 and c-fos expression. Molecularly, p27 and c-fos were direct targets of QKI, while cyclin D1 reduction might be an indirect effect. Taken together, our results reveal that E2F1 directly transcribes QKI, which, in turn, negatively regulates the cell cycle by targeting multiple cell cycle regulators, forming an E2F1-QKI-pRb/E2F1 negative feedback loop.     

Requirement for phosphatidylinositol-3 kinase activity during progression through S-phase and entry into mitosis

Phosphatidylinositol-3 kinase (PI3K) proteins are important regulators of cell survival and proliferation. PI3K-dependent signalling regulates cell proliferation by promoting G1- to S-phase progression during the cell cycle. However, a definitive role for PI3K at other times during the cell cycle is less clear. In these studies, we provide evidence that PI3K activity is required during DNA synthesis (S-phase) and G2-phase of the cell cycle. Inhibition of PI3K with LY294002 at the onset of S-phase caused a 4- to 5-h delay in progression through G2/M. LY294002 treatment at the end of S-phase caused an approximate 2-h delay in progression through G2/M, indicating that PI3K activity functions for both S- and G2-phase progression. The expression of constitutively activated Akt partially reversed the inhibitory effects of LY294002 on mitotic entry, which demonstrated that Akt was one PI3K target that was required during G2/M transitions. Inhibition of PI3K resulted in enhanced susceptibility of G2/M synchronized cells to undergo apoptosis in response to DNA damage as compared to asynchronous cells. Thus, similar to its role in promoting cell survival and cell cycle transitions from G1 to S phase, PI3K activity appears to promote entry into mitosis and protect against cell death during S- and G2-phase progression.     

pRb and E2f-1 in mouse development and tumorigenesis

Our understanding of how RB and E2F-1 function has progressed significantly from the model in which RB negatively regulates expression of genes required for S phase by binding to and inhibiting E2F-1. Both RB and E2F-1 have been shown recently to possess additional properties and mechanisms of regulation relevant to developmental and tumorigenic processes. In particular, it is now realised that RB has E2F-independent tumor suppressor functions which rely upon the ability of RB to induce differentiation. For its part, E2F-1 is unique amongst E2F family members in its capacity to induce apoptosis and this function is clearly relevant to our appreciation of E2F-1 as a conditional tumor suppressor. E2F-1 can induce both apoptosis and S-phase transition and whether E2F-1 acts as an oncogene or a tumor-suppressor gene may depend on the extent to which E2F-1 induces apoptosis as opposed to G1/S transition.