1: Mol Cell Biol. 2004 Apr;24(7):2968-77. Apoptosis associated with deregulated E2F activity is dependent on E2F1 and Atm/Nbs1/Chk2. Rogoff HA, Pickering MT, Frame FM, Debatis ME, Sanchez Y, Jones S, Kowalik TF. Program in Immunology and Virology, University of Massachusetts Medical School, Worcester, Massachusetts 01655, USA. The retinoblastoma protein (Rb)/E2F pathway links cellular proliferation control to apoptosis and is critical for normal development and cancer prevention. Here we define a transcription-mediated pathway in which deregulation of E2F1 by ectopic E2F expression or Rb inactivation by E7 of human papillomavirus type 16 signals apoptosis by inducing the expression of Chk2, a component of the DNA damage response. E2F1- and E7-mediated apoptosis are compromised in cells from patients with the related disorders ataxia telangiectasia and Nijmegen breakage syndrome lacking functional Atm and Nbs1 gene products, respectively. Both Atm and Nbs1 contribute to Chk2 activation and p53 phosphorylation following deregulation of normal Rb growth control. E2F2, a related E2F family member that does not induce apoptosis, also activates Atm, resulting in phosphorylation of p53. However, we found that the key commitment step in apoptosis induction is the ability of E2F1, and not E2F2, to upregulate Chk2 expression. Our results suggest that E2F1 plays a central role in signaling disturbances in the Rb growth control pathway and, by upregulation of Chk2, may sensitize cells to undergo apoptosis. PMID: 15024084 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 2: Mol Cell Biol. 2002 Aug;22(15):5308-18. E2F1 induces phosphorylation of p53 that is coincident with p53 accumulation and apoptosis. Rogoff HA, Pickering MT, Debatis ME, Jones S, Kowalik TF. Program in Immunology and Virology, Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, Worcester, MA 01655, USA. It has been proposed that the E2F1 transcription factor serves as a link between the Rb/E2F proliferation pathway and the p53 apoptosis pathway by inducing the expression of p19ARF, a protein that regulates p53 stability. We find that although p19ARF contributes to p53 accumulation in response to E2F expression, p19ARF is not required for E2F1-mediated apoptosis. E2F1 can signal p53 phosphorylation in the absence of p19ARF, similar to the observed modifications to p53 in response to DNA damage. These modifications are not observed in the absence of p19ARF following expression of E2F2, an E2F family member that does not induce apoptosis in mouse embryo fibroblasts but can induce p19ARF and p53 protein expression. p53 modification is found to be crucial for E2F1-mediated apoptosis, and this apoptosis is compromised when E2F1 is coexpressed with a p53 mutant lacking many N- and C-terminal phosphorylation sites. Additionally, E2F1-mediated apoptosis is abolished in the presence of caffeine, an inhibitor of phosphatidylinositol 3-kinase-related kinases that phosphorylate p53. These findings suggest that p53 phosphorylation is a key step in E2F1-mediated apoptosis and that this modification can occur in the absence of p19ARF. PMID: 12101227 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 3: Ann Oncol. 2001 Jun;12(6):779-85. Rb, mcl-1 and p53 expression correlate with clinical outcome in patients with liver metastases from colorectal cancer. Backus HH, van Riel JM, van Groeningen CJ, Vos W, Dukers DF, Bloemena E, Wouters D, Pinedo HM, Peters GJ. Department of Medical Oncology, University Hospital Vrije Universiteit, Amsterdam, The Netherlands. BACKGROUND: Thymidylate synthase (TS) has been associated with clinical outcome in disseminated colorectal cancer. However, many patients with low TS expression still fail to respond to treatment. Therefore, we studied the cell cycle proteins, Rb, E2F2, Ki67, p21 and p53 and the apoptotic proteins, mcl-1, hax, bcl-xl, bcl-2, Fas receptor, Fas ligand, caspase-3, M30 and PARP as potential predictive factors. PATIENTS AND METHODS: In biopsy specimens of liver metastases from 31 colorectal cancer patients, protein expression was retrospectively determined by immunohistochemistry and related to response to hepatic arterial or intravenous (i.v.) 5-fluorouracil (5-FU) treatment, time to tumour progression (TTP) and overall survival. RESULTS: Expression of both p53 and Rb correlated with survival benefit after 5-FU treatment. A median survival time of 79 weeks was found in patients with high levels of p53 or Rb compared to 36 and 44 weeks for patients expressing low levels of p53 (P = 0.027) or Rb (P = 0.030), respectively. Multivariate analysis showed that p53 was the best predictor of survival independent of sex, age or prior treatment. Following 5-FU hepatic arterial infusion, patients with a high TS expression had a shorter survival time than those with a low expression (P = 0.025). The anti-apoptotic protein mcl-1 was the only factor, which correlated with response to 5-FU treatment. Thirty-five percent of patients with a diffuse mcl-1 expression responded whereas ninety percent of patients with a peri-nuclear expression responded (P = 0.041). CONCLUSIONS: These results indicate that besides TS, also Rb, p53 and mcl-1 are correlated with clinical outcome in patients with liver metastases from colorectal cancer. PMID: 11484952 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 4: Int J Oncol. 2001 Feb;18(2):343-7. Co-expression of E2F-2 enhances the p53 anti-cancer effect in human glioma cells. Mitlianga PG, Kyritsis AP, Gomez-Manzano C, Lemoine MG, Hu M, Liu TJ, Yung WK, Fueyo J. Department of Neuro-Oncology, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA. Gliomas are highly resistant to conventional treatment. Improved knowledge of the molecular defects of glioma cells offers new avenues for the development of gene therapy strategies. Transfer of the p53 gene has proven effective in suppressing proliferation in human glioma cell lines. However, several human glioma cell lines are resistant to p53-induced cell death. The E2F family of transcription factors are pivotal for the regulation of cell-cycle and cell-death related genes in gliomas. In the present study, we sought a more effective strategy for glioma treatment by examining the therapeutic potential of the simultaneous transfer of p53 and E2F-2 to gliomas. Trypan blue cell viability assays and flow cytometric cell-cycle analysis demonstrated that the transfer of both p53 and E2F-2 induced cell death in D-54 MG, a p53-resistant glioma cell line. In addition, transfer of E2F-2 did not interfere with the apoptotic properties of exogenous wild-type p53 in U-251 MG cells. Finally, the expression of E2F-2 in D-54 MG cells suppressed the expression of the apoptotic molecule mdm-2 induced by exogenous p53 in these cells. These results show that co-expression of E2F-2 and p53 enhances the anti-cancer effect of p53 in gliomas. PMID: 11172602 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 5: Invest Ophthalmol Vis Sci. 2000 Dec;41(13):4223-31. Induction of cell cycle entry and cell death in postmitotic lens fiber cells by overexpression of E2F1 or E2F2. Chen Q, Hung FC, Fromm L, Overbeek PA. Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas, USA. PURPOSE. Previous studies have shown that inactivation of the retinoblastoma tumor suppressor protein (pRb) can cause lens fiber cell proliferation and apoptosis. Because pRb is thought to block cell cycle progression by inhibition of E2F transcription factors, experiments were conducted to test whether overexpression of different E2F family members would be sufficient to induce fiber cell proliferation and subsequent apoptosis. The in vivo functions of the transcription factor E2F2 have not previously been analyzed or described in transgenic mice. METHODS. Human E2F1 and E2F2 cDNAs were linked to the alphaA-crystallin promoter. Transgenic mice were generated by microinjection. Changes in cell cycle regulation were assayed by immunohistochemistry for 5-bromo-2'-deoxyuridine (BrdU) incorporation and by in situ hybridization. Cell death was assayed using the TdT-dUTP terminal nick-end labeling (TUNEL) assay. RESULTS. At embryonic day (E)15.5, strong expression of the E2F1 and E2F2 transgenes was detected in lens fiber cells with little or no expression in epithelial cells. BrdU incorporation and TUNEL assays showed that overexpression of either E2F1 or E2F2 in lens fiber cells was sufficient to cause cell cycle entry and subsequent apoptosis. Expression of either E2F1 or E2F2 was sufficient to induce the transcription of cyclins (A2, B1, and E), as well as p53 and Bax in the lens fibercells. CONCLUSIONS. Expression of either E2F1 or E2F2 can induce postmitotic lens fiber cells to re-enter the cell cycle. Inappropriate cell cycle entry is recognized by p53 in each case, and programmed cell death ensues. PMID: 11095619 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 6: Cell Growth Differ. 1998 Feb;9(2):113-8. E2F1-specific induction of apoptosis and p53 accumulation, which is blocked by Mdm2. Kowalik TF, DeGregori J, Leone G, Jakoi L, Nevins JR. Department of Genetics, Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina 27710, USA. Previous work has demonstrated a role for E2F transcription factor activity in the regulation of cell growth during the G0/G1-S phase transition. Indeed, overexpression of E2F proteins, including the E2F1 and E2F2 products, induces DNA synthesis in quiescent fibroblasts. Other experiments have shown that E2F1 expression also induces apoptosis, dependent on p53. Although this could represent a response to aberrant cell cycle progression, we show that only E2F1 induces apoptosis and that this coincides with an ability of E2F1 to induce accumulation of p53 protein. We also find that coexpression of Mdm2, which is known to regulate p53 activity, blocks the E2F1-mediated induction of apoptosis and also blocks the E2F1-mediated accumulation of p53. We propose that E2F1 acts as a specific signal for the induction of apoptosis by affecting the accumulation of p53, which under normal proliferative conditions may be controlled by Mdm2. PMID: 9486847 [PubMed - indexed for MEDLINE] ---------------------------------------------------------------