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Previous Article | Next Article 
Molecular and Cellular Biology, August 2002, p. 5308-5318, Vol. 22, No. 15
0270-7306/02/$04.00+0 DOI: 10.1128/MCB.22.15.5308-5318.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Program in Immunology and Virology,1 Department of Molecular Genetics and Microbiology,2 Department of Cell Biology, University of Massachusetts Medical School, Worcester, Massachusetts 016553
Received 29 October 2001/ Returned for modification 10 December 2001/ Accepted 26 April 2002
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ABSTRACT |
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INTRODUCTION |
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During normal cell proliferation, E2F family members modulate the expression of many genes involved in the transition from G1 to S phase and DNA replication (53). E2F transcriptional activity is regulated by interaction with members of the retinoblastoma (Rb) family of proteins (24). Inactivation of Rb by cellular or viral oncoproteins or loss of Rb will stimulate cells to bypass G0/G1 and enter S phase (17, 19, 50, 52). S-phase induction also occurs when E2F1, E2F2, or E2F3 is exogenously expressed in quiescent cells (15, 35, 40). There is evidence that ectopic expression of various E2F family members can induce both S-phase progression and apoptosis, depending on the levels of expressed cDNA or cell type context (12, 28, 40, 59, 72, 74, 79). However, in fibroblasts, exogenous expression of E2F1, but not E2F2 or E2F3, causes apoptosis (15, 39).
The link between E2F1 and p53-dependent apoptosis has been demonstrated by genetic studies in mice. Apoptosis observed in transgenic mouse models expressing a fragment of the simian virus 40 large-T antigen and in the central nervous system of Rb-/- mouse embryos is p53-dependent and associated with E2F1 (46, 56, 69). Additionally, expression of an E2F1 transgene in the skin of K5E2F1 transgenic mice results in increased proliferation and p53-dependent apoptosis (57). Recent reports have also identified a p53-independent mechanism of E2F1-induced apoptosis that is mediated by the p53 homologue p73 (44, 67). E2F1 is able to directly transactivate the p73 promoter, thereby inducing a cell cycle arrest and apoptosis (33, 67).
E2F1 has been shown to transactivate the expression of the mouse p19ARF and human p14ARF promoters (4, 32, 60). p19ARF encodes a protein that modulates the activity of Mdm2 (58, 77). Mdm2 is an E3-like ubiquitin ligase that regulates the stability of p53 by promoting its degradation by the proteasome (21, 22, 26, 31). By inhibiting Mdm2 activity, p19ARF allows stabilization, activation, and accumulation of p53 protein (58, 77). Thus, it has been hypothesized that p19ARF is a key protein linking the Rb/E2F and p53 pathways (4, 37). One hypothesis for how aberrations in the Rb/E2F pathway are recognized by p53 is that deregulated E2F expression activates p53 by inducing p19ARF.
Although the three E2F proteins that induce S phase (E2F1, E2F2, and to a lesser extent, E2F3) also induce p19ARF expression (15), only E2F1 induces p53-dependent apoptosis in fibroblasts. These observations suggest that there may be different pathways for E2F1 to signal p53-mediated apoptosis and p53 accumulation. We demonstrate that although p19ARF contributes to p53 accumulation, it is not required for E2F1-mediated apoptosis. We also find that E2F1 expression results in the phosphorylation of N-terminal serine residues on p53, similar to the p53 modifications observed in response to DNA damage. Covalent modification of p53 is required for E2F1-mediated apoptosis and occurs independent of p19ARF status. Moreover, E2F2 expression, which does not induce apoptosis in mouse embryo fibroblasts (MEFs), does not lead to the phosphorylation of serine residues on p53 in the absence of p19ARF.
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MATERIALS AND METHODS |
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Adenovirus vectors. Recombinant adenoviruses encoding p53, E2F1, and E2F2 have been described previously (15, 40, 63). An adenovirus encoding an E2F1 DNA binding mutant, E2F1e132 (35), was created by homologous recombination (40, 54). The p53N/C virus was generated from the pCB6 plus p53N/C construct generously provided by Karen Vousden (National Cancer Institute, Frederick, Md.). Control viruses encode either an empty expression cassette or ß-galactosidase. Infection with control virus had no effect on the parameters tested relative to mock infection (data not shown). Viruses were propagated in 293 cells and purified by centrifugation through cesium-chloride gradients. Virus titers were determined by immunohistochemical staining for the adenovirus hexon protein with an anti-adenovirus antibody (Biodesign International) and visualized by using a 3,3'-diaminobenzidine substrate kit from Vector Laboratories (10).
Virus infection. Cells were washed once with phosphate-buffered saline (PBS), and serum-free DMEM containing the virus was added to the cells. Infections were carried out at 37°C in 5% carbon dioxide (CO2) for 1 h (40). The virus inoculum was then removed and replaced with DMEM containing the appropriate concentration of serum, and the mixture was cultured under the conditions described above.
Immunoblot analysis. MEFs were infected with AdCon, AdE2F1, AdE2F1e132, or AdE2F2 at a multiplicity of infection (MOI) of 100, except for lanes marked E2F1low, which were infected at an MOI of 25. Adp53 was infected at an MOI of 10 unless stated otherwise. Whole-cell extracts from MEFs were harvested at 24 h postinfection (hpi). Cells were washed twice with cold PBS and lysed in whole-cell extract buffer (50 mM HEPES [pH 7.9], 250 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 0.1% Nonidet P-40, 1 mM dithiothreitol, 1x mammalian protease inhibitor cocktail [Sigma], 1x phosphatase inhibitor cocktails I and II [Sigma]) by incubation for 30 min on ice. Soluble proteins were separated by centrifugation at 13,000 x g in a microcentrifuge, and supernatants were stored at -70°C. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and transferred to a polyvinylidene difluoride membrane (Millipore) by electroblotting. p19ARF was detected with anti-p19ARF polyclonal antisera (Novus), p53 protein was detected with monoclonal antibody D0-1 or polyclonal antiserum Ab-7 (Oncogene Research Products), phospho-Ser15 and -Ser20 forms of p53 were detected with polyclonal antisera specific for each modification (Cell Signaling Technologies). Transduced p53N/C and wild-type p53 were detected with the monoclonal antibody PAb1801 to compare expression levels (Oncogene Research Products) (see Fig. 7A). Actin was detected with polyclonal antisera I-19 (Santa Cruz Biotechnology, Inc.). Immunoreactive proteins were detected by using a chemiluminescence kit (Amersham) according to the manufacturer's recommendations. Relative changes in the levels of p53 were estimated from scanned images of Western blots with Multianalyst software (Bio-Rad).
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Apoptosis analysis by ELISA. Cells were plated in 24-well plates at a concentration of 1 x 104 cells per well. Virus infections were performed, as described above, 24 h after plating, and caffeine was added at the indicated doses following infection. At 72 hpi, cells were centrifuged at 500 x g for 10 min at 4°C, and lysed, and an enzyme-linked immunosorbent assay (ELISA) was performed as described by the manufacturer (Roche).
Immunohistochemical staining for p53 protein accumulation. Cells were infected with the appropriate adenovirus constructs and immunohistochemically stained for p53 protein as described previously (39). AdCon, AdE2F1, AdE2F1e132 were infected at an MOI of 100. At the time of harvest (48 hpi), cells were washed three times with PBS and then fixed for 15 min each in 3.7% formaldehyde followed by methanol. The cells were then washed in PBS-0.5% Tween 20. Cells were then incubated with anti-p53 monoclonal antibody PAb421 (Oncogene Research Products) in the presence of 1% bovine serum albumin in PBS-0.5% Tween 20 for 45 min at room temperature. The cells were washed three times with PBS-0.5% Tween 20, and the bound antibody was detected with a Vectastain 3,3'-diaminobenzidine substrate kit as described by the manufacturer.
p53 immunofluorescence. Cells were infected with AdCon, AdE2F1, or AdE2F2 at an MOI of 100, and at 24 hpi, the cells were fixed in 3.7% formaldehyde followed by methanol. Fixed cells were incubated with anti-p53 monoclonal antibody pAb421 (Oncogene Research Products) and polyclonal antisera specific for phospho-Ser15 p53 (Cell Signaling Technologies). The bound antibodies were detected with fluorescein isothiocyanate (FITC)- or Rhodamine Red-X-conjugated secondary antibodies (Jackson Immunoresearch Laboratories, Inc., and Southern Biotechnology Associates, Inc.).
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RESULTS |
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2.4-fold) increase in total p53 protein levels in Adp53-infected p19ARF-/- MEFs compared to AdCon infection of the same cell type (Fig. 2B). The basal levels of ectopically expressed p53 were higher in p19ARF-/- MEFs than in wild-type cells. This observation is consistent with the data obtained by immunostaining and immunoblotting for endogenous p53 in p19ARF-/- MEFs (Fig. 2A) (see Fig. 5C), although the difference is exaggerated when cells are infected with Adp53 (Fig. 2B). Others have also reported higher basal levels of p53 in p19ARF-/- MEFs (16). E2F1 expression minimally affected p53 protein levels in Adp53-infected Mdm2-/-/p53-/- MEFs. We suspect that the Mdm2 deficiency elevates baseline levels of p53 in these cells due to an absence or greatly reduced level of p53 ubiquitin ligase activity. E2F1e132 had little or no effect on p53 protein levels in each cell type, consistent with a transcriptional mechanism being at least partly responsible for E2F1-mediated accumulation of p53. The reduced ability of E2F1 to alter p53 levels above those of the controls in p19ARF-/- MEFs, or in MEFs lacking Mdm2 but expressing p53, is consistent with a model in which E2F1 modulates p53 protein levels, in large part, through the p19ARF/Mdm2/p53 pathway. E2F expression leads to the phosphorylation of p53. In response to DNA damage, p53 is activated by covalent modifications, including the phosphorylation at certain serines and acetylation of lysine residues. Of these modifications, phosphorylation of Ser15 on human p53, or Ser18 on mouse p53, is commonly observed in response to DNA damage (3, 7). Given that both DNA damage (37) and E2F1 do not necessarily require p19ARF to activate p53, we determined whether E2F1-mediated activation of p53 function is coincident with p53 phosphorylation. To detect phospho-Ser18, we used an antibody specific for this modification. We find that E2F1 expression, and to a lesser extent, that of E2F2, in wild-type MEFs results in an increase in endogenous p53 protein levels and the phospho-Ser18 form of p53 as detected by immunofluorescence (Fig. 3). When merged, it is apparent that only a subset of the cells with an increase in total p53 has increased levels of the phospho-Ser18 form of p53. Total p53 protein and the phospho-Ser18 form of p53 were present at low levels in cells infected with the control virus (Fig. 3). These data suggest that E2F1 may signal p53 accumulation and p53 phosphorylation by separate pathways.
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We next examined the role of p53 kinases on apoptosis signaling in response to E2F1 expression by using caffeine, a potent inhibitor of several of these kinases. We find that there is a dose-dependent decrease in apoptosis in E2F1-expressing MEFs following treatment with increasing doses of caffeine (Fig. 8). Treatment of cultured cells with caffeine prior to ionizing radiation results in a delayed and attenuated accumulation of p53 protein (38). This defect in p53 accumulation is due to the inhibition of the ataxia telangiectasia mutated (ATM) and the ATM-Rad3-related (ATR) kinases that phosphorylate p53 on serine 15. Inhibition of ATM and ATR will also block subsequent signals that activate the checkpoint kinases CHK1 and CHK2 and perhaps other kinases that phosphorylate p53 on serine 20 (5, 62). The observations that blocking p53 phosphorylation either by mutating potential phosphorylation sites on p53 or treatment with the p53 kinase inhibitor, caffeine, can significantly compromise apoptosis demonstrates that p53 phosphorylation is a critical step in E2F1-mediated apoptosis.
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DISCUSSION |
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It is conceivable that forcing cells into S phase by ectopic E2F expression can induce the apoptotic response. However, it is not likely to simply be ectopic S-phase induction following E2F expression that triggers apoptosis, since both E2F1 and E2F2 can induce S phase at similar efficiencies (15), and we find that E2F2 does not induce apoptosis in MEFs. Moreover, there does not appear to be a specific phase of the cell cycle in which E2F1 induces apoptosis (14, 40). Our findings that ectopic E2F1, but not E2F2, expression results in increased p53 phosphorylation in the absence of p19ARF and that this covalent modification of p53 contributes to E2F1-mediated apoptosis suggest that E2F1 may activate a cellular response similar to DNA damage. Activation of p53 by covalent modification, specifically phosphorylation of serine 15 (serine 18 in the mouse) and serine 20 in response to DNA damage, has been a topic of great interest due to the location of these residues within the Mdm2 interaction domain of p53. Mutational analysis of p53 sites known to be covalently modified, including serine 15 and serine 20, have been inconsistent regarding the requirements for phosphorylation at any one of these sites for p53-dependent apoptosis in response to DNA damage (2, 71, 75). While our study examined the phosphorylation status of two N-terminal residues on p53 known to be phosphorylated following DNA damage, we have not established that either of these modifications are causal in p53-mediated apoptosis, nor have we ruled out the involvement or importance of modification(s) to other residues on p53 following E2F1 expression. However, our finding that apoptosis is reduced when E2F1 is coexpressed with a p53 mutant lacking many of these phosphorylation sites demonstrates a contribution by potential p53 phosphorylation targets to E2F1-induced apoptosis. Additionally, the observation that caffeine inhibits E2F1-mediated apoptosis suggests that the action of one or more p53 kinases is likely to be important for E2F1 signaling. Signaling cascades that activate protein kinases responsible for phosphorylating N-terminal residues on p53 upon DNA damage are well documented (1). Candidate kinases include ATM, ATR, CHK1, and CHK2. ATM and ATR phosphorylate p53 at Ser15 and activate the CHK kinases by phosphorylation (3, 7, 41, 45, 48). Active CHK kinases can then phosphorylate p53 at Ser15 and Ser20 (66).
Recently, ATM/ATR has been shown to phosphorylate the N terminus of E2F1 but not that of E2F2 or E2F3 (43). This observation, together with the data presented here, leads us to speculate that ectopic E2F1 expression or activation of endogenous E2F1 upon phosphorylation by ATM, leads to increases in the activity and, perhaps, levels of one or more of the p53 kinases, which then phosphorylate p53 and promote apoptosis. Given that the E2F1 DNA binding mutant E2F1e132 did not induce p53 phosphorylation or apoptosis, transcriptional activation of one or more p53 kinases might provide a mechanism for E2F1 activation of this pathway. However, overexpression of the ATM/ATR, CHK1, or CHK2 kinases has been found to be insufficient for their activation (45, 48). Therefore, an additional signal(s) may be necessary to stimulate their activity.
It is conceivable that E2F1, E2F2, or E2F3 could signal through the p19ARF/Mdm2 pathway to increase p53 protein levels and that the increased pools of p53 would provide more substrate for the p53 kinases activated by E2F1 (Fig. 9). Thus, the p19ARF/Mdm2 pathway may act both as an attenuator of proliferation by targeting E2F family members for degradation and as an amplifier of a DNA damage signal by increasing pools of p53 available for phosphorylation. The decision to undergo growth arrest or apoptosis would then depend on the cellular context or extent of DNA damage.
Our data implicates p53 phosphorylation as a key step in E2F1-mediated p53-dependent apoptosis. These observations raise the possibility that E2F1 signaling and DNA damage response pathways may converge and involve the same or related kinases to activate p53. Alternatively, E2F1 may contribute to or be a component of the DNA damage pathway. Given these possibilities, it is possible that a role of E2F1 may be to amplify DNA damage signals, resulting in p53-mediated apoptosis. Future studies are needed to test this hypothesis and to define the kinase(s) involved in p53 phosphorylation and the mechanisms by which E2F1 activates them.
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ACKNOWLEDGMENTS |
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This work was supported by a grant from the American Heart Association (no. 9630085N to T.F.K.) and NIH grants CA86038 (to T.F.K.) and CA77735 (to S.J.). H.R. was supported by an NIH training grant (no. 5T32 AI07349). The Flow Cytometry Core Facility is supported by an NIH Center grant (no. DK32520).
The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of the NIH.
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FOOTNOTES |
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