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Cancer Research 68, 5932, July 15, 2008. doi: 10.1158/0008-5472.CAN-07-6373
© 2008 American Association for Cancer Research
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Systems Biology and Emerging Technologies

Noninvasive Assessment of E2F-1–Mediated Transcriptional Regulation In vivo

Parisa Monfared1,4, Alexandra Winkeler1,2,4, Markus Klein1,4, Hongfeng Li1,4, Anke Klose1,4, Marianna Hoesel3, Yannic Waerzeggers1,4, Sigrun Korsching3 and Andreas H. Jacobs1,2,5

1 Laboratory for Gene Therapy and Molecular Imaging at the Max-Planck Institute for Neurological Research with Klaus-Joachim-Zülch-Laboratories of the Max Planck Society and the Faculty of Medicine of the University of Cologne, 2 Center for Molecular Medicine, 3 Institute of Genetics, and 4 Departments of Neurology, University of Cologne, Cologne, Germany; and 5 Klinikum Fulda, Fulda, Germany

Requests for reprints: Andreas H. Jacobs, Laboratory for Gene Therapy and Molecular Imaging, Max Planck Institute for Neurological Research, Gleuelerstr. 50, 50931 Cologne, Germany. Phone: 49-221-4726-310; Fax: 49-221-4726-298; E-mail: Andreas.Jacobs{at}nf.mpg.de.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
Targeted therapies directed against individual cancer-specific molecular alterations offer the development of disease-specific and individualized treatment strategies. Activation of the transcription factor E2F-1 via alteration of the p16-cyclinD-Rb pathway is one of the key molecular events in the development of gliomas. E2F-1 binds to and activates the E2F-1 promoter in an autoregulatory manner. The human E2F-1 promoter has been shown to be selectively activated in tumor cells with a defect in the pRb pathway. Paradoxically, E2F-1 also carries tumor suppressor function. Our investigations focused on analyzing the dynamics of the activity of the E2F-1 responsive element under basal conditions and certain stimuli such as chemotherapy using molecular imaging technology. We constructed a retrovirus bearing the Cis-E2F-TA-LITG reporter system to noninvasively assess E2F-1–dependent transcriptional regulation in culture and in vivo. We show that our reporter system is sensitive to monitor various changes in cellular E2F-1 levels and its transcriptional control of our reporter system to follow the state of the Rb/E2F pathway and the DNA damage–induced up-regulation of E2F-1 activity in vivo. Exposure to 1,3-bis(2-chloroethyl)-1-nitrosourea leads to increased E2F-1 expression levels in a dose- and time-dependent manner, which can be quantified by imaging in vivo, leading to an alteration of cell cycle progression and caspase 3/7 activity. In summary, noninvasive imaging of E2F-1 as a common downstream regulator of cell cycle progression using the Cis-E2F-TA-LUC-IRES-TKGFP reporter system is highly attractive for evaluating the kinetics of cell cycle regulation and the effects of novel cell cycle targeting anticancer agents in vivo. [Cancer Res 2008;68(14):5932–40]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
Molecular imaging is an emerging technology for the noninvasive assessment of disease-specific molecular events, i.e., transcriptional regulation and signal transduction, in the living organism in vivo. Different strategies have been used to assess a disease-specific molecular target or pathway using reporter genes. One strategy is to place the reporter gene under control of a transcriptionally regulated promoter that is active in the molecular pathway of interest.

Rb is a nuclear phosphoprotein and prevents cell cycle progression by binding to E2F transcription family members. Giving the key role of Rb and its pathway members for controlling progression through the cell cycle, it is apparent that alteration of this pathway will cause uncontrolled cell proliferation. In a majority of tumor types, Rb itself and/or the cell cycle regulatory pathway is dysregulated (1–4). Therefore, the Rb pathway represents a promising target for assessment by imaging as well as therapy (5–8).

One of the best-studied Rb-associated proteins is the group of transcription factors known as the E2F family. During tumorigenesis, an effect of the pervasive Rb pathway changes is the loss of Rb binding to E2F, leading to an increase in transcriptionally active or "free" E2F in tumor cells. The abundance of free E2F results in high-level expression of E2F-responsive genes in tumor cells, including the E2F-1 gene itself, resulting in either uncontrolled proliferation or induction of apoptosis (9, 10). The hypothesis for this study was that alterations of free E2F can be noninvasively quantified through the activation of the E2F-1 promoter driving reporter gene expression using molecular imaging technology (11). Moreover, various studies have shown that DNA damage and cellular stress lead to stabilization and accumulation of E2F-1 with E2F-1 contributing as a specific inducer of p53. It has been shown that E2F-1–induced apoptosis can be both dependent and independent of p53 (12–14).

Chemotherapy with the alkylating agent 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) was one of the most commonly used chemotherapeutic agents for patients with gliomas (15). It has been shown that p53-dependent reporter gene expression can be imaged in vivo in response to DNA damage induced by BCNU (16). In this study, we investigated whether we could differentiate various levels of reporter gene expression being under transcriptional control of an E2F-1 promoter in response to BCNU-induced DNA damage. First, we show that BCNU treatment can up-regulate the expression of E2F-1 at the protein level in human U87dEGFR glioma cells. In addition, the expression level of p53 and p21 proteins increased in parallel with the increase in the expression levels of E2F-1. Next, we constructed two self-inactivating retroviral vectors in which the E2F-TA promoter and TA promoter were placed upstream of a LUC-IRES-TKGFP (LITG) reporter system, respectively. We show that different levels of endogenous E2F-1 activity can be quantified noninvasively in vivo in response to BCNU challenge. Moreover, we show that BCNU has a positive effect on cell cycle arrest and induces apoptosis in a dose-dependent manner. We propose that these types of the reporter systems will allow a detailed insight into the kinetics of cell cycle control and for the development of new cell cycle–targeted molecular therapies.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosure of Potential...
 References
 
Cell culture. Human U87dEGFR glioma cells have functional wild-type p53 (kind gift of Dr. W. Cavenee, Ludwig Institute for Cancer Research, San Diego, CA; ref. 17). The amphotrophic retroviral packaging cell line PHOENIX (kind gift of Dr. Nolan, Stanford University, Standford, CA) was used for packaging of retroviral plasmids. The cell lines were grown as monolayer in DMEM (Life Technologies) supplemented with 10% FCS (PAA Laboratories GmbH) and 100 U/mL penicillin and 100 µg/mL streptomycin (PAA Laboratories GmbH). Retrovirally transduced U87dEGFR cells were selected with 500 µg/mL G418 (PAA Laboratories GmbH). U87dEGFR-E2F-TA-LUCIRESTKGFP, U87dEGFR-TA-LUCIRESTKGFP, and U87dEGFR-LUCIRESTKEGFP (see next paragraph) were obtained by selection in culture medium supplemented with 2 µg/mL puromycin (Sigma).

Generation of reporter vectors. The schematic structures of the retroviral vectors (pBABEpuro Moloney murine leukemia virus–based vector backbone; kind gift of Dr.M. Sena-Esteves, Harvard Medical School, Boston, MA) are depicted in Fig. 1 . E2F-TA firefly luciferase cDNA was amplified from pE2F-TA-Luc (test) and TA-Luc (negative control) from pTA-Luc (Clontech) using forward primers 5'-TCTTACTCGAGCTAGCCTTGGCG-3' and 5'-CGGGCTCGAGATCTAGACTCTAGAGGG-3', respectively, and reverse primer 5'-CCGCGGATCCTCTAGAATTACACGGCGA-3'. XhoI (forward primer) and BamHI (reverse primer) sites were incorporated to facilitate cloning of fragments. The PCR products (E2F-TA-luc and TA-luc) were digested with XhoI and BamHI and replaced upstream of the TKGFP gene into similarly cut pBABE-Neo-IRESTKGFP to generate pBABE-E2F-TA-LUCIRESTKGFP and pBABE-TA-LUCIRESTKGFP, respectively. A positive clone was confirmed by restriction enzyme digestion and sequencing. Thereafter, the neomycin resistance gene in these vectors was exchanged to the puromycin resistance gene using SfiI/NheI restriction sites to generate pBABEpuro-E2F-TA-LUCIRESTKGFP and pBABE-TA-LUCIRESTKGFP retroviral vector plasmids. To assess the transcriptional activity of the human E2F promoter and TA promoter, the self-inactivating feature of the vector was provided by a deletion in the 3' long terminal repeat (LTR) enhancer region (U3). During reverse transcription of the retroviral RNA, the inactivated 3'LTR is copied and replaces the 5'LTR, resulting in inactivation of the 5'LTR enhancer sequences.


Figure 1
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  		Figure 1. Structure of the retroviral vector bearing the cis-E2F-TA-LITG reporter system. This vector has a mutation in the 3'LTR that renders the silencing of its promoter activity after duplication as 5'LTR during integration. The expression of the lucIREStkgfp gene is regulated by an artificial promoter containing four copies of the E2F enhancer element, located upstream of the minimal TA promoter, the TATA box from the herpes simplex virus-1 thymidine kinase promoter. Constitutive expression of the puromycin resistance gene (puro) is driven by the SV40 immediate early promoter, allowing for the selection of stably transduced cells.

 
Packaging and transduction of tumor cell lines. The packaging of pBABEpuro derived plasmids bearing Cis-E2F-LUCIRESTKGFP, TA-LUCIRESTKGFP, and LUCIRESTKGFP into retrovirus particles was performed as described previously (18, 19). In brief, PHOENIX cells were grown as monolayers in 60-mm dishes and transfected with vector by the calcium phosphate method. Forty-eight hours after transfection, the retrovirus containing medium was harvested, filtered (0.45 µm), stored in aliquots at –80° for later use or supplemented with polybrene (8 µg/mL; Sigma), and used to infect proliferating human U87dEGFR glioma cells.

Selection of transduced tumor cell clones. To generate stable cell lines, 48 h after infection, U87dEGFR-E2F-TA-LUCIRESTKGFP–, U87dEGFR-TA-LUCIRESTKGFP–, and U87dEGFR-LUCIRESTKGFP–transduced glioma cells were washed, trypsinized, replated (1:10), and propagated under selection with puromycin (2 µg/mL) for 2 wk. To assess Cis-E2F–mediated regulation of the tkgfp fusion gene in transduced cells, the number of enhanced green fluorescent protein (eGFP)-positive cells and the level of eGFP-fluorescence were investigated as described previously (20).

Chemotherapeutic agent. BCNU (Mr 214.06; Bristol-Myers Pharmaceuticals) was prepared by dissolving 100 mg of BCNU in 30 mL of 10% ethanol and was stored at –70°C to ensure drug stability (21). Subsequently, BCNU stock solution was added to culture medium or was injected i.p. into experimental animals to achieve the desired concentrations for experimental use.

Immunoprecipitation. To estimate the amount of endogenous E2F-1 protein after treatment with BCNU, immunoprecipitation was performed. Total cell extracts were prepared 0, 24, and 48 h after BCNU administration. After washing (ice-cold PBS), cells were resuspended in lysis buffer [10 mmol/L Tris (pH 8), 1 mmol/L EDTA, 1 mmol/L DTT, and 0.5% Triton], including proteinase inhibitor mix (Roche Diagnostics GmbH), and incubated (30 min; ice). Samples were cleared by centrifugation, and supernatants were collected. Equal protein amounts were precleared with protein A/G-agarose (Santa cruz) for 1 h at 4°C under gentle rotation. Thereafter, immunoprecipitation was performed by adding monoclonal anti-E2F1 antibody KH95 (Santa Cruz) and protein A/G-agarose at 4°C. Beads were washed thrice (washing buffer), and bound proteins were subjected to Western blot analysis.

Western blot analysis. The expression of E2F-1 in response to BCNU treatment was evaluated by Western blotting. Briefly, monolayers were harvested 24 and 48 h after BCNU treatment and resuspended in PBS. Cells were lysed and the amount of protein present in each sample was quantified using a Bio-Rad protein assay (Bio-Rad Laboratories). Equal amounts of denatured (95°C; 5 min) protein were loaded onto a 10% SDS-polyacrylamide gel. After separation (1 h; 100V) proteins were blotted to a Hybond-ECL membrane (Amersham Pharmacia Biotech) in standard Tris-glycin transfer buffer (100V; 240 mA; 1.25 h). After blocking nonspecific binding (5% nonfat dry milk in 0.1% PBS-Tween20; 1 h), the membrane was washed (0.1% PBS-Tween20) and then probed with primary antibodies anti-E2F KH95 (1:500 dilution), anti-actin C-2 (1:200 dilution; Santa Cruz), respectively, in 5% dry milk and 0.1% PBS-Tween20 (22). After washing, secondary antibodies Goat Anti-Mouse IgG with horseradish peroxides (Southern Biotechnology Associates, Inc.; 1:10,000) were conjugated in 5% dry milk in 0.1% PBS Tween20 for 1 h, and for final protein detection, chemiluminescent reaction (Pierce Biotechnology, Inc.) were used. PCBAS software was used to quantify the intensity of protein expression as assessed by Western blot.

Reverse transcription-PCR and quantitative real-time reverse transcription-PCR. Total RNA was independently extracted from nontreated and BCNU-treated cells using Qiagene RNeasy Plus Mini kit (Qiagene), and 1 µg RNA/sample were reverse-transcribed by SuperScript III reverse transcriptase (200 U; Invitrogen GmbH) using oligo(dT)12–18primers (1 h at 50°C; 15 min at 75°C). Two microliters of U87dEGFR cDNA were subjected to PCR amplification using the HotStar Taq Master Mix kit (Qiagen) with the following specific primers: CD133 forward primer, 5'-CCCTTAATGATATACCTGACAGAG-3'; CD133 reverse primer, 5'-CAAAGACAAAGGTAAGAACCAC-3'. Amplification of NT2D1 cDNA was used as a CD133-positive control. Negative controls were performed by omitting the corresponding cDNA template (Supplementary Fig. S1).

Quantitative reverse transcription-PCR (RT-PCR) was performed using the following specific primers: E2F-1 forward, 5'-CGGCGCATCTATGACA-3'; E2F-1 reverse, 5'-GCAATGCTACGAAGGT-3'; glyceraldehyde-3-phosphate dehydrogenase (GAPDH) forward, 5-'GGTATCGTGGAAGGACT-3'; GAPDH-reverse 5'-GGGTGTCGCTGTTGAA-3'. Real-time PCRs were performed with the LightCycler FastStart DNA MasterPLUS SYBR Green kit using the LightCycler system and normalized to a dilution series of calibrator cDNA using the Relative Quantification Software (Roche Diagnostics) as described (23). Relative quantification was performed by normalized ratio of E2F-1-to-GAPDH expression. Size was verified by melting curve analysis and agarose gel electrophoresis.

Quantification of luciferase gene expression in cell culture. Cells were seeded at a concentration of 5 x 104 cells per well (24-wells). D-luciferin (Synchem) at a concentration of 2 mg/mL were added immediately before assay. Bioluminescence signal was measured using a plate reader (Berthold Tech.) and analyzed by Mikro Win 2000 software.

Immunocytostaining. Cells (2 x 106) were seeded on coverslips placed in 24-wells and incubated for 24 h in DMEM (10% fetal bovine serum; 1% P/S). The following day, cells were washed with PBS (3 x 5 min), fixed with paraformaldehyd (24), and washed with PBS (3 x 5 min). Cells were permeabilized (0.2% Triton X-100 in PBS; ref. 22) and washed with PBS (3 x 5 min). Unspecific binding of antibodies was prevented by blocking cells [3% bovine serum albumin (BSA); 60 min]. Thereafter, cells were washed (0.3% BSA/PBS) and incubated with the first antibody (in 0.3% BSA/PBS; 90 min). Afterwards, cells were washed and incubated for 1 h with the secondary antibody (in 0.3% BSA/PBS) and washed again (PBS x5; bidest x1). Cover slips were removed from 24-wells, put on glass slides, and sealed using Citi Fluor (Citifluor Ltd).

Cell cycle analysis. Cells were plated into 6-cm plates (1 x 106 cells per plate). Cells were treated with different concentrations of BCNU (15–45 µmol/L). At 24 h posttreatment, cells were trypsinized, combined with any floating cells, and pelleted. Cells were washed with PBS twice and repelleted. All centrifugations were at 700 x g for 5 min at 4°C. Subsequently, cells were fixed in ethanol (final concentration, 70%) and stored at –20°C for 4 h. Cells were pelleted and suspended with PBS with RNAase (10 mg/mL). Cellular DNA was stained with propidium iodide (SIGMA-ALDRICH Chemie GmbH; ref. 22). The cellular DNA content was analyzed by flow cytometry (fluorescence-activated cell sorting; FACSCalibur; Becton Dickinson).

Caspase-3/7 activity assay. The activities of caspase-3/7 were measured using the Caspase-Glo 3/7 kit (Promega Corp.). Briefly, cells were grown in 96-well plates until the semiconfluency stage and then treated with the indicated concentrations of BCNU for 24 h in culture medium. Plates were removed from the incubator and kept at room temperature for 30 min, and the Caspase-Glo 3/7 reagent (100 µL) was added. Plates were further incubated for 1 h at 37°C. Luminescence was monitored using a Mithras LB940 plate reader (Berthold Technologies). All experiments were performed in triplicate.

Animal experiments. Animal procedures were performed in accordance with the German laws for animal protection and were approved by the local animal care committee and the Bezirksregierung Köln. Three sets of animals (n = 18 in total) were studied. Each set of animals (n = 6) bearing test (n = 2), negative control (n = 2), and positive control (n = 2) tumor cells was divided into nontreated (n = 3) and BCNU-treated animals (n = 3). The animals were under anesthesia by i.p. injection of ketanest (80 mg/kg) and rompun (16 mg/kg). Tumor cells (1 x 106 cells in 50 µL of DMEM) were injected s.c. into each site in the shoulders and flanks of nude mice as follows: U87dEGFR-E2F-TA-LUCIRESTKGFP (test, n = 2), U87dEGFR-TA-LUCIRESTKGFP (negative control, n = 2), and U87dEGFR-LUCIRESTKGFP (20), giving rise to 4 tumors per animal. On days 7 to 9 posttumor implantation, xenografts reached a size of ~5 mm in diameter, and BCNU (5, 15, 25, and 35 mg/kg body weight) was injected (i.p.) in 50% of animals.

Bioluminescence imaging in culture and in vivo. For the assessment of cells in culture, D-luciferin (Synchem) was added to tissue culture medium to a final concentration of 2 mg/mL. Five minutes later, photons were counted using the IVIS200 imaging system (Xenogen). Data were analyzed using LivingImage software (version 2.50; Xenogen). For in vivo studies, analysis of luciferase gene expression was performed at multiple time points after BCNU administration. For imaging, mice were injected i.p. with D-luciferin (4 mg per animal in 200 µL PBS) and images were acquired 10 min after luciferin injection at standard camera settings with exposure time of 1 to 5 min. Data evaluation was based on a region of interest analysis of bioluminescence imaging images to quantify different signal intensities.

Statistics. Descriptive statistics and regression analysis were performed with Microsoft Excel 2002 (Microsoft Corp). Student's t test as well as Mann-Whitney Rank sum test were performed with SigmaStat 3.0 (SPSS, Inc.). Statistical significance was set at <5% level (P < 0.05).


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosure of Potential...
 References
 
Induction of E2F-1 accumulation in U87dEGFR cells after BCNU treatment. Upon DNA damage, one consequence of a deregulated pRb/E2F pathway is an increase in E2F-1 protein level. To investigate the possible role of E2F-1 in mediating response to BCNU, E2F-1 protein level was analyzed in U87dEGFR cells treated with BCNU (25 µmol/L) for 24 and 48 hours by immunoprecipitation and Western blot. As shown in Fig. 2A , untreated cells show a basal level of E2F-1 expression, which increased after exposure to BCNU with a maximum at 24 hours and a later decrease at 72 hours (Supplementary Fig. S2A). The densitometric quantification of E2F-1 expression as shown in Fig. 2B show the quantitative increase relative to the number of U87dEGFR cells for three independent experiments. These results indicate that BCNU treatment has a significant positive effect on the E2F-1 protein level in U87dEGFR cells (P < 0.001; Student's t test). In contrast, E2F-1 protein was decreased within 24 hours at higher doses of BCNU (35 µmol/L; Supplementary Fig. S2B).


Figure 2
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  		Figure 2. A substantial induction of E2F-1 protein level in response to BCNU treatment. A, immunoprecipitation (IP) of E2F-1 in nontreated and BCNU-treated cells at different incubation times shows a BCNU-induced increased expression of E2F-1. The primary anti–E2F-1 recognizes E2F-1 protein at ~60 kDa. The 55 and 27 kDa heavy and light IgG chains, respectively, of the primary antibody are also detected. B, the E2F-1 protein levels as depicted in A were normalized with respect to cell number and investigated by densitometric analysis. Significant differences between BCNU-treated and nontreated cells (P < 0.001; Student's t test) are indicated by * (P < 0.01) and ** (P < 0.001). Columns, mean; bars, SD. The findings depicted in A and B represent the results of three independent experiments. C, Western blot analysis of p53 and p21 levels in nontreated and BCNU-treated U87dEGFR cells shows increased p53 and p21 expression after BCNU exposure. The level of the actin protein was used as loading control. Note that p53, p21, and actin were recognized by antibodies at ~50, 20, 40 kDa, respectively. D, effect of BCNU on the amounts of E2F-1 mRNA and GAPDH mRNA determined by quantitative RT-PCR in a time-dose–dependent manner. Amplified fragments of E2F-1 cDNA (bottom bands) and GAPDH cDNA (top bands) in an agarose gel stained with ethidium bromide and visualized by UV illumination; GAPDH was used as housekeeping gene.

 
BCNU induces p53 and p21 expression in U87dEGFR cells. DNA damage induces checkpoints to prevent damaged cells from progressing through the cell cycle, and p53 plays an important role in protecting cells from the effects of DNA damage. p21 is transcriptionally activated by p53, and previous studies have described a pathway involving the E2F-1–mediated checkpoint signaling by activation of an Atm/p53/p21 pathway after DNA damage (25, 26). Therefore, we also evaluated p53 and p21 protein level in response to BCNU exposure by Western blot analysis. As shown in Fig. 2C, p53 and p21 levels increased in treated cells compare to nontreated cells with a maximum at 24 and 48 hours after exposure to 25 µmol/L BCNU.

The level of E2F-1 mRNA is not increased upon BCNU treatment. To test whether the increase in E2F-1 protein levels observed upon BCNU treatment is a result of enhanced transcription of the E2F-1 gene, qRT-PCR was performed. Cells were treated with 25 and 35 µmol/L BCNU for 6 and 24 hours. As shown in Fig. 2D, there was no significant change in the level of E2F-1 mRNA in response to BCNU treatment. This result indicates that the BCNU treatment does not predominantly cause an induction of E2F-1 at the transcriptional level.

Vector construction. To determine the activity of the E2F responsive element itself and the induction of E2F-1 mediated by BCNU treatment, three modifications were made. The functional basis of the reporter is shown schematically in Fig. 1 (see also Materials and Methods):

(a) The self-inactivation of the 3'LTR that limits the expression of the virus in transduced cells and also limits expression in the case that recombination should occur (27, 28) through the self-inactivation strategy has been shown to be incomplete for silencing viral expression (29). Because the viral LTR is not used to drive viral expression in the transduced cell, a heterologous internal promoter can be inserted in the transfer vector.
(b) E2F-TA-Luc was inserted into self-inactivating pBABE-IRES-TKGFP vector. E2F-TA-luc contains four copies of the E2F enhancer element, located upstream of the minimal TA promoter. Located downstream of promoter is the firefly luciferase reporter gene (30).
(c) In addition, we also constructed two additional reporters: a negative control, in which TA-luc was placed into the self-inactivating pBABE-IRES-TKGFP vector, and a positive control, in which luciferase cDNA was inserted into pBABE-IRES-TKGFP vector and constitutively expressed.

Characterization of E2F-1 regulated LITG expression in culture. To evaluate whether the Cis-E2F-TA-LITG reporter provides quantitative information on the dynamics of E2F-1 activity, we imaged U87dEGFR-E2F-TA-LITG after treatment with BCNU.

To study drug dose- and time-dependent variations of luciferase gene expression in cell culture, U87dEGFR cell lines stably expressing firefly luciferase were made from different reporter vectors (Fig. 1). Luciferase activity was first monitored using the IVIS imaging system (Fig. 3A ). The light intensity was higher in U87dEGFR stably expressing firefly luciferase under the control of E2F-responsive elements compared with negative control. In BCNU-treated cells, there was an increase in light emission in U87dEGFR-E2F-TA-LITG cells 24 hours after BCNU treatment. There was no change in LUC-signal in negative and positive control cells in response to BCNU treatment (Fig. 3A). For quantitative analysis of different levels of LITG expression in response to BCNU, a microplate reader was used. A 3.6-fold difference was observed in cells carrying the E2F-regulated LITG construct compared with negative control. In these cells, the induction of luciferase expression was increased (3.95-fold) in response to BCNU exposure, whereas no significant increase was observed in negative and positive control cells (Fig. 3B). This observation was found to occur in a BCNU dose-dependent manner (Supplementary Fig. S2C).


Figure 3
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  		Figure 3. Activation of the E2F promoter in response to BCNU treatment in cell culture. A, E2F-responsive transcriptional activity was imaged by the IVIS imaging system. Measurements were performed in nontreated and BCNU-treated U87dEGFR cells stably expressing the LITG construct. An increased luciferase signal was observed only in the E2F-1–regulated cells and not in negative and positive control cells. B, relative E2F-1 transcriptional activity was quantified by a microplate reader. E2F-1–regulated cells showed insignificant higher basal expression of luciferase compared with negative control (*, P < 0.05) and a lower expression compared with positive control (**, P < 0.01). After BCNU treatment, luciferase signal is significantly increased in E2F-1–regulated cells (***, P < 0.001) but not in negative and positive control cells. C, colocalization of LUC and GFP in U87dEGFR cells stably expressing E2F-TA-LITG and in negative and positive control cells. Luciferase expression was determined by anti-LUC.

 
We also investigated how the activity of the green fluorescence gene in Cis-E2F-TA-LITG correlated to E2F-1 activity in cell culture by fluorescence microscopy (Supplementary Fig. S2D). Quantification of the relative green fluorescence signal per single cell using MPI-tool (20) showed a higher level of green fluorescence (1.8-fold) in cells carrying the E2F-regulated LITG construct in response to BCNU treatment compared with nontreated E2F cells (Supplementary Fig. S2E). These observations were confirmed by immunocytostaining of U87dEGFR-E2F-TA-LITG cells. The colocalization of IRES-mediated coregulated LUC and TKGFP protein coexpression in cells carrying the E2F-regulated LITG construct is depicted in Fig. 3C. Taken together, these results indicate that there is a good correlation between the reporter gene and E2F-1 activity.

BCNU treatment leads to cell cycle arrest in a dose-dependent manner. Given the up-regulation of E2F-1 in response to BCNU treatment and a major role of E2F-1 in cell cycle regulation, we evaluated the cell cycle distributions of U87dEGFR-E2F-TA-LITG cells after exposure to different doses of BCNU. As shown in Fig. 4A , 24 hours after exposure with various doses of BCNU, there was a significant increase in S-phase populations in treated cells compared with nontreated cells. In addition, a substantial increase in G1 phase was only observed at the highest does of BCNU. These observations show that BCNU induces a block in the G2 phase.


Figure 4
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  		Figure 4. The effect of BCNU (15–45 µmol/L) on cell cycle progression and caspase-3/7 activity in U87dEGFR-E2F-TA-LITG cells. A, the graphs depict cell cycle distribution of U87dEGFR-E2F-TA-LITG cells. Cell cycle distributions show sustained S-G2 phase arrest after treatment with different concentrations of BCNU up to 35 µmol/L concentration. However, a high BCNU concentration (45 µmol/L) leads to G1 and G2 arrest as indicated by an increased G1 and S-G2 fraction. B, the activities of caspase-3/7 were measured using the Caspase-Glo 3/7 kit in U87dEGFR-E2F-TA-LITG cells cultured in 96-well plates, treated with BCNU (24 h), and subjected to Caspase-Glo 3/7 reagent (100 µL; 1 h) and luminescence recording. BCNU leads to a dose-dependent increase of Caspase 3/7 activity up to a dose of 35 µmol/L. Statistically significant differences were observed for BCNU concentrations ranging from 25 to 45 µmol/L (*, P < 0.05; **, P < 0.01; ***, P < 0.01). Each value represents the average of three different experiments.

 
BCNU treatment induces apoptosis. We further evaluated the effect of BCNU in induction of caspase-mediated apoptosis by measuring the activation of caspases-3/7 activity in U87dEGFR-E2F-TA-LITG cells treated with various doses of BCNU (Fig. 4B). Caspase-3/7 activation was measured after 24 hours in the presence or absence of BCNU. BCNU increased the activities of caspase-3/7 in a dose-dependent manner with highest activation at concentrations between 25 and 45 µmol/L. The caspase-3/7 activity in cells treated with the 45 µmol/L concentration was lower most likely due to the cytotoxic effect of BCNU at this concentration.

Visualization of E2F-1 transcriptional activity in vivo. To access the efficacy of the Cis-E2F-TA-LITG reporter system in vivo, mice were imaged daily after i.p. administration of luciferin (Fig. 5A ). As expected, the nontreated test mice showed a basal intensity of LUC-activity, which was 2.5-fold higher than the background level in negative control mice. In BCNU-treated test mice, a 1.9-fold induction of luminescence intensity was observed 24 hours after treatment. Significant changes were neither found for nontreated test mice nor for negative and positive control mice (Fig. 5B). This was also observed in a dose-response study 24 hours after BCNU treatment (Supplementary Fig. S3A and B). The time-dependent changes of E2F-1 activation are depicted in Supplementary Fig. S3C. Test mice showed a significant increase in luciferase signal 24 hours after BCNU treatment with decreasing signals starting at 72 hours after treatment. These results indicate that the dynamics of activity of endogenous E2F-1 protein and its regulation by an exogenous stimulus in tumor cells can be detected and quantified by in vivo imaging.


Figure 5
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  		Figure 5. BCNU-induced E2F-1 activity as determined in vivo. A, E2F-regulated cells and negative and positive control cells were implanted as a set of four tumors in the back of different groups of experimental mice. Mice were followed over time by bioluminescence imaging until tumors could be clearly visualized. Mice were then subjected to BCNU treatment (50%) or control treatment (50%), and repeat imaging was performed 24 h later. An increased luciferase signal was observed only in mice bearing E2F-1–regulated cells and not in mice bearing negative and positive control cells. Color scale, luminescent signal intensity; blue, least intense signal; red, most intense signal. B, mean of the total bioluminescent signals emitted from E2F-1–regulated tumors and negative and positive control tumors in response to BCNU administration. Columns, mean of three independent experiments with n = 6 animals per group; bars, SD. Significant differences are indicated by * (P < 0.05) and ** (P < 0.05).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosure of Potential...
 References
 
In this study, we show the feasibility of imaging E2F-1–dependent transcriptional activity in culture and in vivo. We describe the development of a recombinant self-inactivating retroviral vector encoding LUCIRESTKGFP and its use to monitor various levels of E2F-1 activity in response to DNA damage. We show that different levels of endogenous E2F-1 activity can be quantified noninvasively in vivo in response to BCNU challenge. To our knowledge, this is the first description of noninvasive monitoring of an increased E2F-1 activity in response to DNA damage.

First, we assessed the basal activity of E2F-1 in human U87dEGFR glioma cells and examined whether the E2F-1 activity is altered in response to BCNU. The substantial basal E2F-1 protein level, which was observed by immunoprecipitation, is most likely free E2F-1 as characteristic feature of a deregulation of the Rb/E2F pathway (Fig. 2A and B). U87dEGFR cells have a wild-type p53 gene with EGFRvIII expression and inactivation of p16 and PTEN. Consistent with this deregulation, these cells have an increase in the level of total pRb protein and mostly in the hyperphosphorylated form (31, 32), which leads to release of E2F-1 and stimulates cells to bypass the G0-G1 checkpoint and enter into S phase. However, this unscheduled entry into S phase can trigger apoptosis when deregulated E2F-1 activity reaches a certain threshold induced by an DNA-damaging agent (33). In U87dEGFR cells, a significant induction of E2F-1 expression after BCNU treatment may also indicate the role of E2F-1 in mediating DNA damage signaling in a time- and dose-dependent manner (Supplementary Fig. S2A and B; Fig. 2A and B).

Several studies indicated that E2F-1 serves as the primary link between loss of Rb-mediated growth control and DNA damage signaling. A report by Kowalik and colleagues (34, 35) showed that E2F-1 signaling through a specific subset of DNA damage response factors activates p53 and kill cells. Recent studies have also shown that E2F-1 and p53 are cooperated in response to DNA damage (25, 26). Our investigation also confirmed that both p53 and later p21 protein levels were increased in parallel with E2F-1 induction by BCNU (Fig. 2C). This may involve a number of parallel and perhaps synergistic mechanisms, and additional studies have to fully elucidate the intricate network by which E2F-1 and p53 work together to regulate the cellular response to DNA damage.

We next revealed that the increase in E2F-1 protein levels observed here is not a result of enhanced transcription of the E2F-1 gene itself rather than a reflection of an increase in E2F-1 protein stability (Fig. 2D). Several reports have described an increase in E2F-1 protein resulting from a stabilization of the E2F-1 protein after the treatment of various tumor cells with DNA damaging agents (12, 36). Other studies also reported that E2F-1 induces apoptosis through transcriptional activation or an increase in unbound E2F-1 itself (37, 38).

Because an increasing number of reports have illustrated the "yin and yang" activity of E2F-1 function with the "yin" in its function as oncogene and the "yang" in its function as tumor suppressor (39–41), we created an E2F-1–regulated expression system to allow the noninvasive monitoring of E2F-1–dependent transcriptional regulation in vivo. Similar to the approach to image p53 transcriptional activity by using a Cis-p53TKGFP reporter system (16), we constructed and tested a derivative of a self-inactivating retroviral vector bearing an E2F-TA promoter (Fig. 1). The E2F promoter encompasses E2F binding sites (11, 42). This promoter responds to the activity of Rb in that loss of Rb function releases E2F-1 from its bound state and subsequently activates its own E2F promoter in a positive feedback loop. In agreement with our results from immunoprecipitation, we show that the Cis-E2F-TA-LITG construct was sufficiently sensitive to monitor up-regulation of E2F-1 expression after treatment with BCNU in stably transduced human U87dEGFR glioma cells in culture (Supplementary Fig. S2; Figs. 2A–B and 3). These results are in accordance with reports demonstrating that E2F-responsive promoters are active in glioma cells because of an excess of free E2F as a result of loss of Rb/E2F repressor complexes (9, 43, 44).

We further investigated the effects of our BCNU-challenging system on the cell cycle distribution and caspase activity. G2 arrest occurs in various cell types in response to nitrosoureas (45, 46) including BCNU (15) and other alkylating agents (45). BCNU caused two different types of growth inhibition in our experiments. At low concentrations (15–35 µmol/L), we observed an accumulation of cells in S-G2 phase and a decrease in the G1 population suggesting mitotic arrest. At a higher concentration (45 µmol/L), rapid accumulation of cells in G1 phase occurred within 24 hours (Fig. 4A). The BCNU dose-dependent effects on the cell cycle indicate different response mechanisms mediating either cell cycle arrest to allow for DNA repair or induction of apoptosis (Fig. 4B). Treatment with low concentrations of BCNU (15 µmol/L) caused growth inhibition and only minor caspase-3/7 activity, whereas treatment with high concentrations caused significant caspase 3/7 activity. Our findings are in keeping with those of Xu and colleagues (47) who observed that after 30 h BCNU treatment, there was an increase in S phase population in U87MG cells compared with nontreated cells. Resistance to BCNU in U87MG cells with intact p53 function was associated with an up-regulation of p53, prolonged induction of p21, sustained cell cycle arrest, and a significant enhancement of DNA repair. In contrast, Batista and colleagues (48) showed that BCNU treatment induced a late apoptotic response in U87MG cells. We conclude that S phase arrest in U87dEGFR glioma cells may occur in two steps, first mediated by E2F-1/p53 cooperation, thereafter, the sustained arrest in S phase mediated by p53 and p21. The decision to undergo growth arrest or apoptosis depends most likely on the BCNU dose-dependent extent of DNA damage.

Clinical dosages of BCNU are often limited by toxicity to other organ systems, and the actual drug concentrations delivered to glioma sites in vivo may be lower than the levels that induce apoptosis and cell death. Therefore, we next investigated whether the Cis-E2F-TA-LITG reporter system could be used to quantitate E2F-1 activity within a glioma in response to various doses of BCNU in vivo (Supplementary Fig. S3; Fig. 5). Analysis of signal intensities at different time points showed that 25 mg/kg BCNU treatment were effective in inducing a signal starting at 24 hours, which decreased again after 72 hours.

Several investigators have shown that variation in multidrug resistance genes (i.e., DNA repair activity) have been speculated to cause BCNU chemoresistance in glioblastoma multiforme (GBM; ref. 49). More recent evidence indicated the presence of CD133+ brain tumor stem–like cells within human GBM that may account for this heterogeneity (50). We also observed that U87dEGFR cells contained a subpopulation of CD133+ cells (Supplementary Fig. S1). It has also been reported that the CD133+ subpopulation of GBM is selectively resistant to radiation and chemotherapy (49).

It should be pointed out that recent studies have used E2F-1 apoptotic target genes as new therapeutic strategy or drug targets, thereby providing insight into the basic mechanisms of E2F-1–induced apoptosis and its possible clinical implications. A study by Fine and colleagues (11) described an E2F-responsive adenoviral vector for brain tumors that promises to target cancer cells more specifically than the standard approach, thereby reducing the toxicity to normal tissue of the therapy.

In conclusion, we show that our Cis-E2F-TA-LITG reporter system can monitor the dynamics of E2F-1–dependent gene expression in vivo. This type of molecular imaging paradigm shall help to further elucidate the in vivo dynamics of the complex E2F pathway, its interrelation with other cell cycle regulating proteins, as well as its effect on cellular growth, death, and response to various stimuli, such as DNA damage.


   Disclosure of Potential Conflicts of Interest
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
References
 
No potential conflicts of interest were disclosed.


   Acknowledgments
 
Grant support: Deutsche Forschungsgemeinschaft (DFG-Ja98/1-2), Center for Molecular Medicine Cologne (CMMC-TV46), 6th FW EU grant European Molecular Imaging Laboratories (LSHC-CT-2004-503569), Diagnostic Molecular Imaging (LSHB-CT-2005-512146), and CLINIGENE (LSHB-CT-2006).

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.

We thank G. Schneider for the excellent technical assistance.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

Received 11/24/07. Revised 4/24/08. Accepted 5/12/08.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 

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