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[Cancer Research 61, 6693-6697, September 15, 2001]
© 2001 American Association for Cancer Research

Advances in Brief

Transfer of E2F-1 to Human Glioma Cells Results in Transcriptional Up-Regulation of Bcl-21

Candelaria Gomez-Manzano, Paraskevi Mitlianga, Juan Fueyo, Ho-Young Lee, Min Hu, Kevin B. Spurgers, Tricia L. Glass, Dimpy Koul, Ta-Jen Liu, Timothy J. McDonnell and W. K. Alfred Yung2

Departments of Neuro-Oncology [C. G-M., P. M., J. F., M. H., D. K., T. L. G., T-J. L., W. K. A. Y.], Thoracic and Head and Neck Medical Oncology [H-Y. L.], and Molecular Pathology [K. S., T. J. M.], The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, Texas 77030


   ABSTRACT
Top
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 
Strong evidence exists to support the tenet that activation of E2F transcription factors, via alterations in the p16-cyclin D-Rb pathway, is a key event in the malignant progression of most human malignant gliomas. The oncogenic ability of E2F has been related to the E2F-mediated up-regulation of several proteins that positively regulate cell proliferation. However, E2F may indirectly enhance proliferation by activating antiapoptotic molecules. In this work, we sought to ascertain whether E2F-1-mediated events involve the up-regulation of the antiapoptotic molecule Bcl-2. Western blot analyses showed up-regulation of Bcl-2 but not of Bcl-xL by 24 h after the transfer of E2F-1. Northern blot studies showed that transfer of E2F-1 also up-regulated Bcl-2 RNA. In support of these findings and the concept that E2F-1 has a direct effect in the induction of Bcl-2, we found a putative E2F binding site within the Bcl-2 sequence. Subsequent gel-mobility shift and supershift experiments involving the CTCCGCGC site in the bcl-2 promoter showed that E2F-1 bound Bcl-2. Transactivation experiments consistently showed that ectopic E2F-1 activated responsive elements located in the -1448/-1441 region in the P1 promoter region of the bcl-2 gene. As expected, other members of the E2F family of transcription factors such as E2F-2 and E2F-4 also transactivated the bcl-2 promoter. Our results demonstrate that E2F-1 modulates the expression of the antiapoptotic molecule Bcl-2 and suggest that up-regulation of Bcl-2 may favor the oncogenic role of E2F-1 and other members of the E2F family of transcription factors.


   Introduction
Top
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 REFERENCES
 
The oncogenic property of E2F-1 is thought to be related to its ability to regulate the expression of genes critical for cell proliferation. In support of the tenet that E2F-1 can behave as an oncogene, the p16-Rb pathway, which negatively regulates the ability of E2F-1 to transactivate cell-cycle and DNA-replication-related genes, is partially inactive in the vast majority of cancer cells including most malignant gliomas (reviewed in Ref. 1 ). Moreover, transfer of E2F-1 to quiescent fibroblasts results in entry into the cell cycle and eventually replication of DNA (2) . In addition, transgenic overexpression of E2F-1 favors the formation of skin tumors in vivo (3) . However, inconsistent with its role as an oncogene, E2F-1 also induces programmed cell death in vitro and in vivo (4 , 5) . The ability of E2F-1 to induce either cell proliferation or cell death can be tissue-specific; transgenic ablation of the expression of the E2F-1 gene in mice resulted in the spontaneous generation of tumors in certain tissues and atrophy and lack of proliferation in others (6 , 7) . E2F-1-mediated proliferation is connected to the direct activation of positive regulators of cell cycle- and DNA-replication enzymes. The cancer phenotype is characterized by abrogation of apoptosis as well as unregulated proliferation. It seems plausible that E2F-1 would protect proliferation by activating antiapoptotic genes. However, very few genes have been discovered that are both purely antiapoptotic and controlled by E2F-1.

The Bcl-2 protein is one of the best known antiapoptotic molecules. The bcl-2 gene was isolated from a common human follicular B-cell lymphoma (8) . Rather than inducing aberrant proliferation, like the vast majority of oncogenes, Bcl-2 extends the life span of B cells by suppressing apoptosis (9) . The relationship between Bcl-2 and the acquisition of an antiapoptotic phenotype has been demonstrated in null Bcl-2 and transgenic Bcl-2 animal models; Bcl-2-deficient mice showed abundant lymphocyte cell death (10) . In addition, gain of function of Bcl-2 in mice renders their thymocytes resistant to apoptosis (11) . The connection between E2F-1 and Bcl-2 was established after the discovery that Bcl-2 can protect cells from E2F-1- mediated apoptosis (12 , 13) .

We undertook this work to ascertain whether E2F-1 can activate the expression of Bcl-2. We found that E2F-1 can up-regulate the expression of Bcl-2 at the protein and mRNA levels. We also identified an E2F binding site in the promoter region of Bcl-2 and showed that E2F-1 protein binds this E2F site. We additionally demonstrated that E2F-1, E2F-2, and E2F-4 can transactivate responsive Bcl-2 elements. This report links for first time the regulation of Bcl-2 to E2F-1 transactivation function. Our results suggest that activation of antiapoptotic genes, in addition to up-regulation of positive modulators of cell cycle progression and DNA replication, may favor the oncogenic function of E2F-1.


   Materials and Methods
Top
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 REFERENCES
 
Cell Lines and Culture Conditions.
The U-251 MG, U-87 MG, and T98 G cell lines were obtained from the American Type Culture Collection (Manassas, VA). All of the cell lines were maintained in DMEM/F12 medium (1/1, v/v) supplemented with 10% heat-inactivated FCS in a humidified atmosphere containing 5% CO2 at 37°C. Synchronization procedures are described elsewhere (14) . Briefly, T98 G cells were serum-starved for 3 days by culturing in MCDB-105 serum-free medium (Sigma Chemical Co., St. Louis, MO.) and then stimulated into synchronous cell cycling progression by replacing the medium with DMEM containing 10% FCS.

Adenoviral Vectors and Infection Conditions.
The generation and characterization of the recombinant-deficient adenovirus vectors carrying E2F-1 and the virus control Ad5CMV-pA have been described in detail elsewhere (5) . The adenoviral vectors carrying the cDNA of E2F-2 or E2F-4 (15) were the generous gift of Dr. Joseph R. Nevins (Duke University Medical Center, Durham, NC). Cell lines were cultured and infected as reported previously (16) . We used a multiplicity of infection (defined as the ratio of the number of infectious virions to the number of susceptible cells) of 100.

Immunoblotting Assay.
Total cell lysates were prepared by incubating cells for 1 h at 4°C in radioimmunoprecipitation assay buffer [150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 20 mM EDTA, and 50 mM Tris (pH 7.4)] at different times after infection. Then, 20 µg of protein from each sample was subjected to 7% or 15% SDS-Tris-glycine gel electrophoresis and transferred to a nitrocellulose membrane (Schleicher & Schuell Inc., Keene, NH). The membrane was blocked with Blotto-Tween [3% nonfat milk, 0.05% Tween 20, 0.9% NaCl, and 50 mM Tris (pH 7.5)] and incubated with the following primary antibodies: mouse anti-E2F-1 (KH95; Santa Cruz Biotechnology Inc., Santa Cruz, CA), rabbit anti-E2F-2 (C-20; Santa Cruz Biotechnology Inc.), rabbit anti-E2F-4 (C-108; Santa Cruz Biotechnology Inc.), mouse anti-Bcl-2 (C-2; Santa Cruz Biotechnology Inc.), mouse anti-Bcl-x (PharMingen, San Diego, CA), mouse antihuman p53 (D0–7; DAKO), and mouse antihuman actin IgG (Amersham Corp., Arlington Heights, IL). The secondary antibodies were horseradish peroxidase-conjugated antirabbit IgG, antimouse IgG (both from Amersham), and antigoat IgG (Santa Cruz Biotechnology Inc.). The membranes were developed according to Amersham’s enhanced chemiluminescence protocol.

Northern Blotting.
U251 MG cells (5 x 106) were seeded onto a 10-cm plate and allowed to adhere overnight. The next day, the cells were infected with Ad5CMV-E2F-1, AdE2F-2, AdE2F-4, or Ad5CMV-pA at a dose of 100 multiplicity of infection. The total cellular RNA was isolated 36 h after infection by the acid-guanidium thiocyanate method. For the Northern blotting, 15 µg of total cellular RNA prepared from each sample were subjected to electrophoresis on a 1% agarose gel containing 2% formaldehyde, stained with ethidium bromide, photographed, transferred to a nylon membrane (Zetaprobe; Bio-Rad Laboratories, Hercules, CA), and hybridized to an [{alpha}-32P]dCTP-labeled Bcl-2 cDNA probe. Random priming was performed with the Prime It kit (Stratagene, La Jolla, CA), after which the membrane was washed in high-stringency conditions and autoradiographed for 24–48 h.

Nuclear Extracts and Electrophoretic Gel Mobility Shift Analysis.
U-251 MG cells (106 cells in 100-mm dishes) were washed with PBS and resuspended in 400 µl of hypotonic buffer [20 mM HEPES (pH 7.9), 10 mM KCl, 0.2 mM EDTA, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride, 0.5 µg/ml leupeptin, and 0.5 µg/ml aprotinin] and incubated at 4°C for 15 min. Cells were then lysed by adding 0.1% NP40 and vortexing; nuclei were pelleted and resuspended in 200 µl of hypertonic buffer [20 mM HEPES (pH 7.9), 400 mM NaCl, 10 mM EDTA, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride, 0.5 µg/ml leupeptin, and 0.5 µg/ml aprotinin]. After thorough mixing on a rotating wheel at 4°C for 15 min, the suspension was centrifuged at 14,000 x g for 5 min. The supernatant was used as the nuclear extract for the gel mobility shift assay.

Bandshift assay were performed as follows: nuclear extracts (5 µg) were preincubated with 2 mg of the polydeoxynucleotide poly-dI:dC for 15 min at 4°C and then incubated with labeled oligomer DNA for 15 min at 4°C in the presence of 10 mM Tris-HCl (pH 7.5), 10 mM KCl, 1 mM EDTA, 20% glycerol, 1 mM DTT, and 5 mM MgCl2. An oligonucleotide (25-bp) containing the E2F(a) binding site (in capital letters; Ref. 14 ; sense: 5'atttaagCTCCGCGCcctttctcaa3'; antisense: 3'taaattcGAGGCGCGggaaagagtt5') were synthesized (Life Technologies, Inc., Rockville, MD), annealed to each other, and forward-labeled with a [{gamma}-32P]ATP using T4 polynucleotide kinase. For competition experiments, cold wild-type E2F(a) or mutant E2F(a) [mE2F(a), similar to E2F(a) but with a mutation in the binding site: sense: CTCCGATC; antisense: GAGGCTAG] oligonucleotides (10 x or 100 x molar excess) were mixed with the reaction mixtures before addition of the labeled probe. Reaction products were separated on 5% nondenaturing polyacrylamide gel (38:2 acrylamide:bis-acrylamide) in Tris-glycine electrophoresis buffer for 2 h at 4°C at 180 V. The gel was then dried and autoradiographed with an amplified screen at -80°C. For antibody supershift experiments, nuclear extracts were preincubated with 4 µl of high-concentration anti-E2F-1 antibody (Geneka Biotechnology Inc., Montreal, Quebec, Canada) for 1 h on ice before the addition of labeled oligonucleotide probe.

Plasmid Constructs.
Bcl-2 promoter luciferase constructs were generated using the three-step cloning strategy described previously (17) . Briefly, a 308-bp product was PCR amplified with primers generating XhoI and HindIII sites from a 7.8-kb genomic HindIII fragment of the bcl-2 gene. This 308-bp product covers a region 5' to the AccI site that is just 5' of the initiation codon. This product was cloned into the XhoI and HindIII sites of the pGL3 basic luciferase vector (Promega Corp., Madison, WI) producing pGL3–308 bcl-2. Next, a 486-bp XhoI, AccI restriction fragment was cloned into pGL3–308 bcl-2 producing pGL3–748 bcl-2. Finally, a 2100-bp XhoI fragment was cloned into pGL3–748 bcl-2 producing pGL3–2.8 bcl-2. (Promoter sequence of bcl-2: GenBank accession nos. X51898 and M13994).

Luciferase Reporter Assays.
U-251 MG cells were seeded in 6-well dishes at a density of 5 x 105 cells/well and cultured for 24 h in DMEM/F12 medium containing 10% FCS. Cells were then cotransfected with 5 µg of pGL3–2.8 Bcl-2-Luc, pGL3–748 Bcl-2-Luc, or pGL3–308 Bcl-2-Luc constructs and with 1 µg of pRL-CMV (containing the cDNA encoding Renilla luciferase) by using the FuGENE 6 transfection reagent (Roche Diagnostics Corp., Indianapolis, IN). Cells were infected with Ad5CMV-E2F-1, AdE2F-2, AdE2F-4, or AdCMV-pA 1 h later. After 24 h, lysates were collected and assayed for luciferase activity by using the dual-luciferase reporter assay system (Promega). Luciferase activity from untreated control cells was used for the background signal. All of the firefly luciferase values were normalized to the Renilla luciferase readout values and expressed as x-fold induction relative to that of Ad5CMV-pA-infected cells.

Luciferase reporter assays were also performed with the E2F-1 expression vector pXCJL-E2F-1 (18) . This plasmid contains an E2F-1 expression cassette comprising the human cytomegalovirus promoter E2F-1 cDNA and the SV40 early polyadenylation signal. A similar construct, pXCJL-CMV-pA, lacking the E2F-1 cDNA was used as a control.

Flow Cytometric Analyses of Cell Cycle.
To measure DNA content, 106 cells were trypsinized, fixed in 70% cold ethanol, and incubated with propidium iodide (5 mg/ml) and RNase A (1 mg/ml) for 20 min at 37°C. All of the measurements were made with an EPICS profile flow cytometer (Coulter Corp., Hialeah, FL) equipped with an air-cooled argon ion laser emitting 488 nm at 15 mW. Multicycle (Phoenix Flow System, San Diego, CA) program was used for data analysis.


   Results
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 REFERENCES
 
Cell Cycle Analyses and Expression of Endogenous E2F-1 and Bcl-2.
Because E2F activity plays a major role in inducing cell progression from G1 to S phase, we assessed whether the up-regulation of E2F correlated temporally with up-regulation of the endogenous Bcl-2 protein in T98 G cells as follows. T98 G cells were made quiescent by serum starvation and then stimulated with serum and harvested at different time points from 0 to 28 h (Ref. 14 ; Fig. 1Citation ). After serum stimulation, we observed a more or less synchronized cell-cycle progression with a predominant G0/G1 phase at 0 h, a progressive accumulation of cells in the S phase of the cell cycle between 16 and 20 h, and an increased presence of the number of cells in the G2-M phase by 24 h. As expected, in the Western blot analyses, we detected an increase in the expression level of the endogenous E2F-1 protein 12 h after serum stimulation, immediately before cells accumulated in S phase, and continued increase up until 24 h (exit of cells from S phase). The expression level of Bcl-2 protein began to increase in parallel with the increase in the expression level of E2F-1. These results suggested that the expression of E2F-1 and Bcl-2 molecules might be connected.



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  		Fig. 1. E2F-1 and Bcl-2 expression during synchronized cell-cycle progression. For this experiment cells were harvested at the same times for both flow cytometric analyses and immunoblot assays (see "Material and Methods" and Ref. 14 ). A, the flow cytometric plots display the cell-cycle distribution of quiescent T98 G glioma cells after serum stimulation. The axis represent number of cells on the ordinate and DNA content on the abscissa; B, Western blots show the expression of the endogenous E2F-1 and Bcl-2 proteins at the indicated points after serum stimulation. The level of the protein actin is showed as a loading control. Note that, as expected, increase in level of expression of the E2F-1 protein correlated temporally with cell entry into S phase.

 
Transfer of E2F-1 to Glioma Cells Enhances Expression of Bcl-2.
Next, we investigated whether overexpression of E2F-1 could up-regulate Bcl-2 in human glioma cells. Analysis of protein extracts from E2F-1-transduced U-251 MG cells, growing asynchronously, revealed large increases in Bcl-2 protein levels (Fig. 2)Citation . Consistent with the previous analyses of the endogenous E2F-1 and Bcl-2 expression in a synchronized population of human glioma cells, in these experiments, up-regulation of Bcl-2 was evident by 16 h after transfer of an exogenous E2F-1 and increased progressively, achieving their maximum by 24–48 h after infection (Fig. 2)Citation . Interestingly, extending the Western blot analyses to include Bcl-xL showed that the transfer of E2F-1 did not up-regulate Bcl-xL, a member of the Bcl-2 family of proteins, with antiapoptotic activity. The E2F-1-mediated up-regulation of Bcl-2 was also observed in U-87 MG cells. Moreover, transduction of U-251 MG and U-87 MG cells with adenoviral vectors carrying E2F-2 or E2F-4, two other members of the E2F family of transcription factors of which their DNA-binding domains share a high homology with that of E2F-1, also produced up-regulation of Bcl-2 (Fig. 2)Citation . These results suggest that the E2F family of transcription factors was involved in the modulation of Bcl-2 expression in these gliomas cells.



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  		Fig. 2. Western blot analyses of Bcl-2 protein expression in human glioma cells expressing exogenous E2F-1, E2F-2, or E2F-4. A, Bcl-2, Bcl-xL, and p21 protein levels expressed by U-251 MG glioma cells after infection with adenovirus carrying the E2F-1 coding sequence, assayed at the indicated time points. The up-regulation of Bcl-2 in these cells was evident within 16 h after infection with Ad5CMV-E2F-1. Transfer of E2F-1 did not result in an increased level of expression of Bcl-xL, another antiapoptotic molecule. As described previously (14) , p21 protein levels increased after E2F-1 transfer. B and C, Bcl-2 protein levels expressed by U-251 MG (B) and U-87 MG (C) glioma cells 48 h after infection with adenoviruses carrying the E2F-1, E2F-2, and E2F-4 coding sequences or with the adenovirus control (CMV). Level of expression of actin is shown as a loading control.

 
E2F-1 Up-Regulates Bcl-2 RNA.
Because E2F-1 is a transcription factor, we hypothesized that E2F-1-mediated up-regulation of Bcl-2 should be associated with a concomitant increase in bcl-2 gene expression. Northern blot analyses of U-251 MG cells infected with adenovirus constructs containing E2F-1, E2F-2, or E2F-4 revealed increases in Bcl-2 RNA levels 36 h after infection (Fig. 3)Citation , indicating that E2F-1 either transcriptionally activated Bcl-2 or stabilized Bcl-2 RNA.



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  		Fig. 3. Up-regulation of bcl-2 by E2F. A, Northern blot of Bcl-2 RNA expression in U-251 MG cells 36 h after infection with Ad5CMV-E2F-1, AdE2F-2, AdE2F-4, or control (CMV) constructs, or medium only (Mock). Consistent with the Western blot analyses, transfer of any of the three members of the E2F family of transcription factors resulted in up-regulation of Bcl-2 RNA. B, verification that equal amounts of total RNA had been examined. Ethidium bromide staining of the agarose gel is presented as loading control.

 
Identification of a Putative E2F Binding Site in the Human Bcl-2 Promoter.
The presence of E2F-responsive elements in the bcl-2 promoter would be a strong indication that E2F regulates Bcl-2 at a transcriptional level. When we used the consensus E2F binding sequence (TTTCGCGC) to seek sequence homology within the bcl-2 gene sequence, we found no homologous E2F sites in the bcl-2 promoter. However, when we examined the bcl-2 promoter sequence using another E2F binding sequence (CTCCGCGC) [E2F(a)] present in the human p21/cip1 promoter (14) , we identified a 100% homologous sequence in the -1448/-1441 P1 region of the bcl-2 promoter.

E2F Proteins Bind to the Putative E2F Site.
After identifying the putative E2F binding site in the bcl-2 promoter, we confirmed that nuclear extract-derived proteins could form DNA-protein complexes with the E2F probe (Fig. 4)Citation . In competition experiments with nuclear extracts from U-251 MG human glioma cells, binding of the cellular proteins to the E2F probe was competed out by the unlabeled wild-type probe but not by the unlabeled mutated probe. Addition of an anti-E2F-1 antibody supershifted the complex, indicating that the E2F-1 protein was forming part of the complexes (Fig. 4)Citation .



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  		Fig. 4. E2F-1 binding to the bcl-2 promoter. Electrophoretic mobility-shift assays were performed with a radiolabeled 25-bp oligonucleotide containing the E2F-1 binding region [E2F(a)] (Lane 1, labeled oligonucleotide probe only). U-251 MG nuclear extracts were mixed with the labeled E2F(a) probe (Lanes 2 and 7) or, for competition assays, with labeled E2F(a) and a 10- or 100-fold excess of unlabeled probe [E2F(a)] (Lanes 3 and 4) or mutant probe lacking E2F binding activity [mE2F(a)] (Lanes 5 and 6). *supershift caused by addition of an antibody to E2F-1 (Lane 8).

 
The Bcl-2 Promoter Is Regulated by E2F.
Although these findings provide strong evidence that E2F-1 transcriptionally regulates bcl-2, the ultimate test is to show that the exogenous E2F-1 protein can transactivate a chimeric construct encompassing the putative responsive elements of the E2F protein in the bcl-2 promoter and a reporter gene. For these experiments, we transfected U-251 MG glioma cells with one of three plasmid constructs, each containing different lengths of the bcl-2 promoter linked to a luciferase reporter gene: pGL3–2.8 bcl, pGL3–748 bcl-2, or pGL3–308 bcl-2. After transfection, the cells were infected with Ad5CMV-E2F-1 and examined for luciferase activity 24 h later. Cells that had been transfected with pGL3–2.8 bcl-2, which contained both the P1- and P2-responsive elements, showed 70.8 ± 22 times the activity of the bcl-2 promoter as that of the control (Fig. 5)Citation . In contrast, no significant induction of luciferase activity was noted in cells that had been transfected with the shorter constructs that contained only the P2-responsive elements (Fig. 5)Citation . To ascertain whether the up-regulation of bcl-2 was attributable to the adenoviral-mediated high level of expression of the exogenous E2F-1, we performed a similar luciferase assay transiently transfecting glioma cells with the plasmid construct pXCJL-E2F-1. These experiments showed that cells that were cotransfected with pGL3–2.8 bcl-2 displayed an increase in the luciferase activity that was 17.2, 4, and 3.4 times higher than that of the pXCJL-CMV-pA-transfected cells in three independent experiments. These results suggest that E2F-1 transactivates the bcl-2 promoter through the CTCCGCGC site located in the P1 promoter region. Transfer of E2F-2 or E2F-4 also increased luciferase activity but, again, only in cells that had been transfected with the bcl-2 construct that contained both the P1 and P2 regions. These results strongly suggest that the sequence CTCCGCGC located in the -1448/-1441 position of the bcl-2 promoter is an E2F binding site.



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  		Fig. 5. E2F-1 transactivates Bcl-2-responsive elements. A, schematic representation of Bcl-2-luciferase reporter constructs. Note that three constructs comprise the P2 region but only the pGL3–2.8 bcl2 construct encompasses the P1 region. B, U-251 MG glioma cells were cotransfected with the bcl-2 reporter constructs and the pRL-CMV vector, and 1 h later the cells were treated with adenoviral vectors carrying E2F-1, E2F-2, E2F-4, or an empty expression cassette (CMV). Luciferase activity was determined 24 h after the infection. Nontransfected cells were used as the background. All values were normalized for expression of Renilla luciferase, which served as internal control for transfection efficiency and expressed as x-fold induction relative to that of the adenovirus control-infected cells (equal to 1). Each experiment was performed at least three times in duplicate. Shown are means of normalized luciferase measurements; bars, ± SE. The E2F-mediated induction of the bcl-2 responsive elements within the pGL3–2.8 bcl-2 construct was at least 30-fold higher than the control-mediated induction.

 

   Discussion
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
REFERENCES
 
Our results showed that the adenoviral transfer of E2F-1 to glioma cells led to the up-regulation of Bcl-2, which suggests that E2F-1 favors proliferation through the induction of antiapoptotic genes in addition to being a positive regulator of cell cycle- and DNA replication-related genes. The region within the bcl-2 promoter that was responsive to E2F-1 was found to be in the P1 promoter region (17 , 19) . Electrophoretic mobility assays showed that the E2F-1 protein interacted directly with the E2F binding site. Moreover, an exogenous wild-type E2F-1 was able to up-regulate a reporter gene driven by the bcl-2 promoter. Evidence that other members of the E2F family of transcription factors, like E2F-2 and E2F-4, also modulated the expression of Bcl-2 suggests that the up-regulation of Bcl-2 is not part of a nonspecific reaction of the cells to the overexpression of exogenous E2F-1. The hypothesis that E2F-1 specifically up-regulates Bcl-2 is also supported by our finding that E2F-1 did not up-regulate other members of the Bcl-2 family of proteins such as Bcl-xL. Consistent with these results, Dong et al. (20) reported finding increased levels of the Bcl-2 protein, assessed by Western blotting, after adenovirus-mediated E2F-1 transfer in a melanoma cell line. In addition, Müller et al. (21) , using microarrays technology, showed that retrovirally mediated transfer of E2F-1 to U2OS cells resulted in up-regulation of Bcl-2.

We showed here that E2F-1 transcriptionally activates bcl-2. The expression of pro- and antiapoptotic molecules is regulated by multiple mechanisms. For example, the Bcl-2 family is regulated by cytokines and other death-survival signals at different levels. Some of these signals involve post-translational modification or conformational changes. In addition, several members of the Bcl-2 family of proteins are transcriptionally regulated. For example, Bax seems to be transcriptionally activated by p53, because Bcl-xL and Bcl-2 are also transcriptionally responsive (reviewed in Ref. 22 ). In this regard, the transcriptional regulators of Bcl-2 that have been identified include the p53 tumor suppressor gene product, the products of the cellular and viral myb genes, and the product of the Wilms’ tumor gene wt1 (23) .

Overexpression of Bcl-2 is known to block E2F-1-induced apoptosis (12 , 13) . In the system described by Strom et al. (12) , overexpression of E2F-1 overrode the survival functions provided by granulocyte colony-stimulating factors and trigger apoptosis. However, coexpression of both E2F-1 and Bcl-2 resulted in the acquisition of an antiapoptotic phenotype. Lind et al. (13) reported that cells lacking bcl-2 expression respond to growth factors withdrawal by liberating E2F-1 from inactive complexes resulting in cell death. Another aspect of the interplay between E2F-1 and Bcl-2 is the ability of Bcl-2 to retard the entry of cells into S phase (24 , 25) . Vairo et al. (26) reported that Bcl-2 expression delays E2F-1 accumulation during G1 progression. These authors suggest that gain of E2F-1 function would diminish the Bcl-2 cycle-inhibitory activity and potentially enhance its oncogenic impact. Taken collectively, these observations and our data imply the existence of a feedback regulatory loop that controls the expression and cell effect of E2F-1 and Bcl-2.

We have shown previously that transfer of E2F-1 to glioma cells resulted in apoptosis (5) . In this report, the experiments were performed under similar conditions to those reported previously (5) . The up-regulation of Bcl-2 (antiapoptotic molecule and negative regulator of cell cycle) by E2F-1 (positive regulator of cell and proapoptotic) seems to be counterintuitive. However, it is often the case for transcription factors involved in cancer that produce both positive and negative survival signals. In this regard, c-myc and E1A are able to induce proliferation and apoptosis signals. Importantly, the regions of E1A and c-myc that are responsible for transformation are also necessary for induction of apoptosis, indicating that the growth-promoting activities of c-myc and E1A are linked to its death-inducing properties (recently reviewed in Ref. 27 ). Transfer of p53 to cancer cells results in the up-regulation of pro-apoptotic molecules such as Bax, but also in the up-regulation of molecules that negatively regulates its apoptotic function, such as hdm-2 (28) . The up-regulation of Bcl-2 is not the only paradoxical effect of the transfer of E2F-1 to cancer cells. E2F-1 induces the expression of several genes related to cell-cycle progression but, at the same time, up-regulates proteins that negatively influence cell cycle progression, including p18INK4c (21) . Because transfer of E2F-1 results in the production of several lines of decision, the fate of an E2F-1-treated cell may rely on the status and expression of other genes. For instance, the level of E2F-3 may be important in the proliferation/apoptosis decision-making process. Thus, it has been postulated that E2F-1 and E2F-3 contribute to a pool of free E2F activity that activates inappropriate proliferation once it reaches one critical threshold level (proliferation threshold) but apoptosis once it exceeds a second, higher threshold level (29) . The "free" E2F-3 (like the free E2F-1) activity is arguably high in cancer cells with abnormally regulated retinoblastoma pathway, playing a role in the neoplastic phenotype. Increasing the level of E2F-1 in this background will surpass the proliferative threshold and trigger apoptosis. Finally, despite the fact that overexpression of bcl-2 also follows retroviral- (21) and plasmid-mediated transfer of E2F-1, we cannot completely rule out the possibility that the induction of bcl-2 is only seen because of the use of an adenoviral system where E2F can be highly overexpressed.

The full spectrum of E2F target genes remains to be determined. The E2F-1 up-regulation of Bcl-2 is consistent with the up-regulation of other molecules with antiapoptotic activities such as p21 (16 , 30) . Interestingly, the p21 and bcl-2 promoters share the same E2F-1 binding site (14) . Because expression of these two molecules may enhance the resistance of cells to apoptosis and, therefore, favor the ability of E2F-1 to behave as an oncogene, it is relevant to mention that Bcl-2 and p21 are overexpressed in the majority of malignant gliomas (31 , 32) . In this regard, it will also be interesting to see whether tumors generated by the transgenic expression of E2F-1 overexpress Bcl-2 and p21. Because E2F-1 is able to induce apoptosis, this hypothesis also predicts that the apoptosis observed in the null-Bcl-2 mice might be attributable, at least in part, to E2F-1.


   ACKNOWLEDGMENTS
 
We thank Polly Y. Lee (Department of Neuro-Oncology, The University of Texas M. D. Anderson Cancer Center) for technical assistance and Christine Wogan (Department of Scientific Publications, The University of Texas M. D. Anderson Cancer Center) for editorial assistance.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by NIH Grants RO1 CA83127 (to W. K. A. Y.), RO1 CA80748 (to J. F.), and PO1 CA78778 (to T. J. M.). Back

2 To whom requests for reprints should addressed, at Department of Neuro-Oncology, Box 100, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: (713) 794-1285; Fax: (713) 794-4999. Back

Received 11/29/00. Accepted 7/26/01.


   REFERENCES
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 

  1. Alemany R., Gomez-Manzano C., Balague C., Yung W. K. A., Curiel D. T., Kyritsis A. P., Fueyo J. Gene therapy for gliomas: molecular targets, adenoviral vectors, and oncolytic adenovirus. Exp. Cell Res., 252: 1-12, 1999.[Medline]
  2. Johnson D. G., Schwarz J. K., Cress W. D., Nevins J. R. Expression of transcription factor E2F1 induces quiescent cells to enter S phase. Nature (Lond.), 365: 349-352, 1993.[Medline]
  3. Pierce A. M., Fisher S. M., Conti C. J., Johnson D. G. Deregulated expression of E2F1 induces hyperplasia and cooperates with ras in skin tumor development. Oncogene, 16: 1267-1276, 1998.[Medline]
  4. Wu X., Levine A. J. p53 and E2F-1 cooperate to mediate apoptosis. Proc. Natl. Acad. Sci. USA, 91: 3602-3606, 1994.[Abstract/Free Full Text]
  5. Fueyo J., Gomez-Manzano C., Yung W. K. A., Liu T. J., Alemany R., McDonnell T. J., Shi X., Rao J. S., Levin V. A., Kyritsis A. P. Overexpression of E2F-1 in glioma triggers apoptosis and suppresses tumor growth in vitro and in vivo. Nat. Med., 4: 685-690, 1998.[Medline]
  6. Field S. J., Tsai F. Y., Kuo F., Zubiaga A. M., Kaelin W. G., Jr., Livingston D. M., Orkin S. H., Greenberg M. E. E2F-1 functions in mice to promote apoptosis and suppress proliferation. Cell, 85: 549-561, 1996.[Medline]
  7. Yamasaki L., Jacks T., Bronson R., Goillot E., Harlow E., Dyson N. J. Tumor induction and tissue atrophy in mice lacking E2F-1. Cell, 85: 537-548, 1996.[Medline]
  8. Tsujimoto Y., Gorham J., Cossman J., Jaffe E., Croce C. M. The t(14;18) chromosome translations involved in B-cell neoplasms result from mistakes in VDJ joining. Science (Wash. DC), 229: 1390-1393, 1985.[Abstract/Free Full Text]
  9. Hockenbery D., Nunez G., Milliman C., Schreiber R. D., Korsmeyer S. J. Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death. Nature (Lond.), 348: 334-336, 1990.[Medline]
  10. Veis D. J., Sorenson C. M., Shutter J. R., Korsmeyer S. J. Bcl-2-deficient mice demonstrate fulminant lymphoid apoptosis, polycystic kidneys, and hypopigmented hair. Cell, 75: 229-240, 1993.[Medline]
  11. Sentman C. L., Shutter J. R., Hockenbery D., Kanagawa O., Korsmeyer S. J. bcl-2 inhibits multiple forms of apoptosis but not negative selection in thymocytes. Cell, 67: 879-888, 1991.[Medline]
  12. Strom D. K., Cleveland J. L., Chellappan S., Nip J., Hiebert S. W. E2F-1 and E2F-3 are functionally distinct in their ability to promote myeloid cell cycle progression and block granulocyte differentiation. Cell Growth Differ., 9: 59-69, 1998.[Abstract]
  13. Lind E. F., Wayne J., Wang Q. Z., Staeva T., Stolzer A., Petrie H. T. Bcl-2-induced changes in E2F regulatory complexes reveal the potential for integrated cell cycle and cell death functions. J. Immunol., 162: 5374-5349, 1999.[Abstract/Free Full Text]
  14. Hiyama H., Iavarone A., Reeves S. A. Regulation of the cdk inhibitor p21 gene during cell cycle progression is under the control of the transcription factor E2F. Oncogene, 16: 1513-1523, 1998.[Medline]
  15. DeGregori J., Leone G., Miron A., Jakoi L., Nevins J. R. Distinct roles for E2F proteins in cell growth control and apoptosis. Proc. Natl. Acad. Sci. USA, 94: 7245-7250, 1997.[Abstract/Free Full Text]
  16. Gomez-Manzano C., Fueyo J., Kyritsis A. P., McDonnell T. J., Steck P. A., Levin V. A., Yung W. K. Characterization of p53 and p21 functional interactions in glioma cells en route to apoptosis. J. Natl. Cancer Inst., 89: 1036-1044, 1997.[Abstract/Free Full Text]
  17. Young R. L., Korsmeyer S. J. A negative regulatory element in the bcl-2 5'-untranslated region inhibits expression from an upstream promoter. Mol. Cell. Biol., 13: 3686-3697, 1993.[Abstract/Free Full Text]
  18. Hunt K. K., Deng J., Liu T. J., Wilson-Heiner M., Swisher S. G., Clayman G., Hung M. C. Adenovirus-mediated overexpression of the transcription factor E2F-1 induces apoptosis in human breast and ovarian carcinoma cell lines and does not require p53. Cancer Res., 57: 4722-4726, 1997.[Abstract/Free Full Text]
  19. Seto M., Jaeger U., Hockett R. D., Graninger W., Bennett S., Goldman P., Korsmeyer S. J. Alternative promoters and exons, somatic mutation and deregulation of the Bcl-2-Ig fusion gene in lymphoma. EMBO J., 7: 123-131, 1988.[Medline]
  20. Dong Y. B., Yang H. L., Elliott M. J., Liu T. J., Stilwell A., Atienza C., Jr., McMasters K. M. Adenovirus-mediated E2F-1 gene transfer efficiently induces apoptosis in melanoma cells. Cancer (Phila.), 86: 2021-2033, 1999.[Medline]
  21. Müller H., Bracken A. P., Vernell R., Moroni C., Christians F., Grassilli E., Prosperini E., Vigo E., Oliner J. D., Helin K. E2Fs regulate the expression of genes involved in differentiation, development, proliferation, and apoptosis. Genes Dev., 15: 267-285, 2001.[Abstract/Free Full Text]
  22. Gross A, McDonnell J. M., Korsmeyer S. J. Bcl-2 family members and the mitochondria in apoptosis. Genes Dev., 13: 1899-1911, 1999.[Free Full Text]
  23. Smith M. D., Ensor E. A., Coffin R. S., Boxer L. M., Latchman D. S. Bcl-2 transcription from the proximal P2 promoter is activated in neuronal cells by the Brn-3a POU family transcription factor. J. Biol. Chem., 273: 16715-16722, 1998.[Abstract/Free Full Text]
  24. O’Reilly L. A., Huang D. C., Strasser A. The cell death inhibitor Bcl-2 and its homologues influence control of cell cycle entry. EMBO J., 15: 6979-6990, 1996.[Medline]
  25. Linette G. P., Li Y., Roth K., Korsmeyer S. J. Cross talk between cell death and cell cycle progression: BCL-2 regulates NFAT-mediated activation. Proc. Natl. Acad. Sci. USA, 93: 9545-9552, 1996.[Abstract/Free Full Text]
  26. Vairo G., Soos T. J., Upton T. M., Zalvide J., DeCaprio J. A., Ewen M. E., Koff A., Adams J. M. Bcl-2 retards cell cycle entry through p27Kip1, pRB relative p130, and altered E2F regulation. Mol. Cell. Biol., 20: 4745-4753, 2000.[Abstract/Free Full Text]
  27. Breckenridge D. G., Shore G. C. Regulation of apoptosis by E1A and Myc oncoproteins. Crit. Rev. Eukaryot. Gene Expr., 10: 273-280, 2000.[Medline]
  28. Oren M. Regulation of the p53 tumor suppressor protein. J. Biol. Chem., 274: 36031-36034, 1999.[Free Full Text]
  29. Ziebold U., Reza T., Caron A., Lees J. A. E2F3 contributes both to the inappropriate proliferation and to the apoptosis arising in Rb mutant embryos. Genes Dev., 15: 386-391, 2001.[Abstract/Free Full Text]
  30. Polyak K., Waldman T., He T. C., Kinzler K. W., Vogelstein B. Genetic determinants of p53-induced apoptosis and growth arrest. Genes Dev., 10: 1945-1952, 1996.[Abstract/Free Full Text]
  31. Alderson L. M., Castleberg R. L., Harsh G. R., IV, Louis D. N., Henson J. W. Human gliomas with wild-type p53 express bcl-2. Cancer Res., 55: 999-1001, 1995.[Abstract/Free Full Text]
  32. Jung J. M., Bruner J. M., Ruan S., Langford L. A., Kyritsis A. P., Kobayashi T., Levin V. A., Zhang W. Increased levels of p21WAF1/Cip1 in human brain tumors. Oncogene, 11: 2021-2028, 1995.[Medline]



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