ARTICLE

Expression of Transcription Factor E2F1 and Telomerase in Glioblastomas: Mechanistic Linkage and Prognostic Significance

Marta M. Alonso, Juan Fueyo, Jerry W. Shay, Kenneth D. Aldape, Hong Jiang, Ok-Hee Lee, David G. Johnson, Jing Xu, Yasuko Kondo, Takao Kanzawa, Satoru Kyo, B. Nebiyou Bekele, Xian Zhou, Janice Nigro, J. Matthew McDonald, W. K. Alfred Yung, Candelaria Gomez-Manzano

Affiliations of authors: Departments of Neuro-Oncology (MMA, JF, HJ, O-HL, JX, WKAY, CG-M), Pathology (KDA, JMM), Neurosurgery (YK, TK), and Biostatistics (BNB, XZ), University of Texas M. D. Anderson Cancer Center, Houston, TX; Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, TX (JWS); Department of Carcinogenesis, University of Texas M. D. Anderson Cancer Center, Science Park-Research Division, Smithville, TX (DGJ); Department of Obstetrics and Gynecology, Kanazawa University School of Medicine, Ishikawa, Japan (SK); Department of Pathology, University of California School of Medicine, San Francisco, CA (JN)

Correspondence to: Candelaria Gomez-Manzano, MD, Department of Neuro-Oncology, Box 1002, University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030 (e-mail: cmanzano{at}mdanderson.org).


    ABSTRACT
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 Notes
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Background: Several tumor suppressor pathways have been identified as modulators of telomerase function. We examined the functional role of the retinoblastoma-E2F1 pathway in regulating telomerase activity in malignant gliomas. Methods: Adenovirus vectors were used to transfer cDNAs into human glioblastoma and sarcoma cells. Telomerase activity was assessed with a telomere repeat amplification protocol. Promoter activity in cancer cells was assessed with promoter–luciferase reporter constructs. Promoter binding was assessed with the chromatin immunoprecipitation (ChIP) assay. We isolated astrocytes from E2F1 transgenic mice and normal mice for in vivo studies. We evaluated the expression of E2F1 and hTERT (the catalytic subunit of human telomerase) mRNAs by reverse transcriptase–polymerase chain reaction and proteins in human glioblastoma samples by immunoblot analysis. Associations between survival among 61 glioblastoma multiforme patients and expression of E2F1 and hTERT mRNA and protein were examined with Kaplan–Meier analysis, the log-rank test, and Cox proportional hazards regression models. All statistical tests were two-sided. Results: Ectopic E2F1 expression increased hTERT promoter activity in cancer cells. We detected an interaction between E2F1 protein and the hTERT promoter. Transgenic E2F1 astrocytes contained functional telomerase protein. E2F1 mRNA expression and hTERT mRNA expression were statistically significantly correlated in human glioblastoma specimens (R = .8; P<.001). Longer median survival was statistically significantly associated with lower E2F1 mRNA expression in tumors (103.6 weeks) rather than with higher expression (46.1 weeks) (difference = 57.5 weeks; 95% confidence interval [CI] = 14.7 to 159.7; log-rank P = .002). E2F1 mRNA was the only factor that was statistically significantly associated with overall survival in a multivariable model (P = .04). Among 27 patients with glioblastoma multiforme samples, the expression of E2F1 protein was statistically significantly associated with survival (log-rank P<.001). Conclusions: E2F1 may participate in telomerase activity regulation in malignant glioma cells. Its expression appears to be strongly associated with the survival of patients with malignant brain tumors.



    INTRODUCTION
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 Notes
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Mechanistic studies of cancer cells at the molecular level may help to elucidate tumor behavior, to predict prognosis, and to develop targeted and efficacious therapies. Transformation of cultured primary cells into tumorigenic variants is a multistep process in which each genetic change confers a proliferative advantage (1). In most cancers, gene mutations disrupt normal cell cycle checkpoints that otherwise limit cell division in response to DNA damage or oncogene activation. The retinoblastoma protein (pRb) is a common downstream target of the signal cascade that regulates cell cycle checkpoints. Particular components of the retinoblastoma (Rb) regulatory network may act as tumor suppressors or proto-oncogenes, mutations in which are apparently essential for cancer cell development (2,3). Inactivation of Rb expression in cancer cells leads to the deregulated activity of the transcription factors E2F1, E2F2, and E2F3, which control the expression of genes involved in differentiation, development, proliferation, and apoptosis. E2F1 has oncogenic properties in vitro and in vivo, and its overexpression is sufficient to induce the transformation of rodent primary cells (4) and to promote tumorigenesis in transgenic mice (5). Surprisingly, E2F1 also induces apoptosis in vitro and in vivo (6), and E2F1-null mutant mice develop spontaneous malignancies, suggesting that E2F1 may be a tumor suppressor (7,8). Thus far, however, E2F1 inactivation or loss-of-function mutations have not been consistently found in human cancer.

A current tenet of carcinogenesis is the hypothesis that human cells must overcome at least two barriers before efficient cellular proliferation can occur. The first barrier, senescence, minimally involves the Rb tumor-suppressor pathway. However, to sustain immortalization, cells must eventually overcome the second proliferative blockade, known as crisis (9,10). To overcome crisis, telomeric DNA must be stabilized through telomerase activation (11) or through activation of the alternative lengthening of telomeres pathway (12,13). Human telomerase is a reverse transcriptase that contains an RNA moiety, known as hTR, and a catalytic subunit, known as hTERT (14,15). hTERT adds telomeric repeats to telomeres by using the RNA moiety hTR as a template (16). In the absence of telomerase activity, telomeric DNA increasingly erodes with each round of cell division (17), so that telomeres progressively shorten, which eventually leads to senescence in normal cells (18). The lack of telomerase activity may also result in end-to-end fusion of chromosomes, chromosome instability, and cell death (19,20).

Several tumor suppressor pathways have been identified as modulators of telomerase function. In addition, recent studies performed in normal human cells found an association between telomerase activity and the cell cycle (21). Current research is focusing on the role of E2F1 as a key regulator of telomerase expression in normal cells (2123), specifically, on the proximal region of the hTERT promoter sequence that contains, at least, three putative E2F1 binding sites (22,23).

We examined the functional role of E2F1 in regulating telomerase in malignant brain tumors. Our primary goal was to determine whether there is a direct link between E2F1 and telomerase biology. If successful, our study would indicate that the Rb-E2F1 pathway is one of the critical tumor-suppressor/oncogene pathways involved in regulating telomerase expression and activity in glioblastoma. An additional goal was to determine whether the overexpression of E2F1 and telomerase might serve as prognostic markers in patients with glioblastoma.


    PATIENTS AND METHODS
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 Notes
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Cell Culture

Human cell lines Saos-2 (osteosarcoma), HeLa (cervical adenocarcinoma), and U-251 MG (malignant glioma) were obtained from the American Type Culture Collection (Manassas, VA). Cells were synchronized as described (24). Briefly, confluent cultures of Saos-2 cells were serum-starved for 3 days by culturing in McCoy's 5a medium (Gibco, Carlsbad, CA) supplemented with 0.1% fetal bovine serum (Gemini, Woodland, CA) and then synchronized by subculturing at a ratio of 1:2 in medium containing 15% fetal bovine serum.

Adenoviral Vectors, Wild-Type Adenovirus, and Infection Conditions

The replication-deficient adenoviral vectors Ad5CMV-E2F1 (Ad-E2F1, where CMV is cytomegalovirus), Ad5CMV-Rb (Ad-Rb), Ad5CMV-p21 (Ad-p21), and Ad5CMV-{beta}-gal (Ad-{beta}-gal, where {beta}-gal is {beta}-galactosidase) and the wild-type adenovirus serotype 5 (Ad-WT) were as described previously (2527). We used a multiplicity of infection of 100 plaque-forming units per cell. As controls, we used the replication-deficient adenoviral vector Ad5CMV-pA (Ad-CMV) (with an empty expression cassette), the wild-type adenovirus inactivated by exposure to seven cycles of 125-J UV radiation (254 nm) (UV-inactivated Ad-WT), and mock infections with culture medium.

Cell Cycle Analysis

The distribution of cells in the cell cycle was analyzed by measuring their DNA content with flow cytometry, as described previously (28). Cell samples were collected at the indicated times after infection with Ad-E2F1, Ad-Rb, Ad-p21, Ad-CMV, Ad-WT, or UV-inactivated Ad-WT or treatment with the cyclin-dependent kinase inhibitor olomoucine (1 µM).

Luciferase Assays

Cells were cultured at a density of 3 x 104 cells per well in 24-well dishes for 24 hours and then transfected with 250 ng of the hTERT promoter–luciferase reporter construct (pGL3-hTERT). To determine transfection efficiency, cells were transfected with 1 ng of the renilla vector pRL-CMV (Promega, Madison, WI) (29). All transfections used the FuGENE 6 transfection reagent (Roche Diagnostics Corp., Indianapolis, IN). One hour after transfection, cells were infected with Ad-E2F1, Ad-Rb, Ad-p21, Ad-CMV, Ad-{beta}-gal, Ad-WT, or UV-inactivated Ad-WT at a multiplicity of infections of 100 plaque-forming units, or cells were treated with 1 µM olomoucine. Cells were harvested 24 hours after treatment, and hTERT promoter–driven luciferase reporter activity was measured via the Dual Luciferase assay (Promega). Luciferase activity from untreated control cells was used as the background signal, normalized by renilla activity, and expressed as the fold of induction relative to mock-treated cells, to which we assigned an arbitrary value of 1 U.

Telomere Repeat Amplification Protocol

We harvested 106 U-251 MG or Saos-2 cells by trypsinization, and the pellet was resuspended in 100 µL of ice-cold CHAPS lysis buffer {0.5% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate [CHAPS], 0.1 mM benzamidine, 10 mM Tris-HCl at pH 7.5, 1 mM MgCl2, 1 mM EGTA, 10% glycerol, and 5 mM 2-mercaptoethanol}, and then RNase inhibitor (Q-Biogene, Irvine, CA) was added to a final concentration of 150 U/mL. Telomerase activity was assayed by using the Chemicon TRAPeze telomerase detection kit according to the manufacturer's protocol (Chemicon, Temecula, CA). This kit is a modification of the telomere repeat amplification protocol (TRAP) developed by Kim et al. (30). Each analysis included a sample incubated at 95 °C for 10 minutes before the assay as a negative control and HeLa cells as a positive control. Every sample contained control primers and a telomere substrate control for amplification of a 36-base-pair (bp) internal standard (internal control). The products of the TRAP assay were resolved by polyacrylamide gel electrophoresis in nondenaturing 12% gels in Tris-glycine buffer (25 mM Tris and 192 mM glycine at pH 7.5). The gel was stained with Syber green (Molecular Probes, Eugene, OR) and visualized at a wavelength of 254 nm in a Fluor-S Multimager (Bio-Rad Laboratories, Hercules, CA). For every TRAP reaction, the relative signal intensity of the band ladder corresponding to multimers of the telomere repeats was measured with the National Institutes of Health (NIH) Image analysis program (developed at the U.S. NIH and available on the Internet at http://rsb.info.nih.gov/nih-image/), and the results were normalized to those from the internal control.

Immunoblot Analysis of TERT and E2F1 Expression

For immunoblot analysis of TERT expression, nuclear extracts were prepared from 80% confluent cell cultures by use of a nuclear extraction kit (Active Motif, Carlsbad, CA) and the manufacturer's instructions. Samples containing identical amounts of protein (20–100 µg, depending on the antibody used) were subjected to sodium dodecyl sulfate–Tris-glycine gel electrophoresis and transferred to nitrocellulose membranes, as described elsewhere (28). Blots were incubated with the antibodies against E2F1 (product KH-95) and {beta}-actin (product C-11) from Santa Cruz Biotechnology (Santa Cruz, CA) and hTERT (product Est-21-A) and mouse TERT (mTERT; product Est-23A) from Alpha Diagnostic (San Antonio, TX). The secondary antibodies were horseradish peroxidase–conjugated anti-mouse, anti-rabit, and anti-goat immunoglobulin G (IgG) antibodies (Santa Cruz Biotechnology). Immunoblots were developed according to Amersham's enhanced chemiluminescence protocol (Amersham Bioscience, Piscataway, NJ). Protein expression was quantified by densitometry analysis with the public domain NIH Image program as explained above.

E2F1 protein levels were analyzed by immunoblot analysis from 27 snap-frozen glioblastoma multiforme specimens (World Health Organization [WHO] grade IV astrocytoma), from patients selected as described below. Signal intensity was normalized to that of tubulin (anti-human tubulin monoclonal antibody T5168; Sigma Chemical Corp., St Louis, MO) levels of expression in every sample. Normal human astrocyte cultures were used as the control for E2F1 expression in normal cells.

Reverse Transcriptase–Polymerase Chain Reaction

Analysis of hTERT, mouse (m) TERT, hTR, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression was performed by reverse transcriptase (RT)–polymerase chain reaction (PCR) amplification. Total RNA was collected from samples with a kit from Quigen (Valencia, CA) by following the manufacturer's protocol. hTERT, mTERT, and hTR mRNAs were amplified with primers and PCR conditions as described elsewhere (3133). Reaction products were then resolved by electrophoresis on 2% agarose gel. Amplification of GAPDH RNA was used as the loading control to normalize the amount of input RNA.

Chromatin Immunoprecipitation Assays

Cells were infected with Ad-E2F1 or Ad-CMV or were mock-infected for 24 hours. The chromatin immunoprecipitation (ChIP) assay was performed by use of the ChIP assay kit (Upstate Biotechnology, Lake Placid, NJ) by following the manufacturer's instructions. E2F1 antibodies (product KH-95) or mouse IgG antibodies (Santa Cruz Biotechnology) were used to immunoprecipitate the cross-linked chromatin. The hTERT proximal promoter was amplified by PCR as described elsewhere (22).

For the ChIP assay to assess mTERT (GenBank accession number AF121949), we designed the following three pairs of primers: 5'-CAAATGCTAGGTCGGTGGAT-3' and 5'-GATGGCAGCTCTGCTAGGTT-3' to generate a product of 273 bp; 5'-CCCTGGCCTCATGTCCTCTAA-3' and 5'-CCCCAAAGAAACCAGCATAG-3' to generate a product of 382 bp; and 5'-CCCTGGCCTCATGTCCTCTAA-3' and 5'-TTTGGCAAACACTGAAATGC-3' to generate a product of 767 bp. PCR conditions were as follows: one cycle at 94 °C for 3 minutes; 33 cycles at 94 °C for 30 seconds, 53 °C for 30 seconds, and 72 °C for 20 seconds; and a 1-minute cycle at 72 °C.

Construction of the Glial Fibrillary Acidic Protein (GFAP)-E2F1 Transgene and Generation of Transgenic GFAP-E2F1 Mouse Lines

The PstI-BamHI fragment from pSI (Promega), including the chimeric intron, multiple cloning region, and simian virus 40 late polyadenylation signal, was inserted into the PstI-BamHI site of pBluescript II KS+ (Stratagene, La Jolla, CA) to produce pBSI. The blunt-ended full-length human E2F1 cDNA was inserted into the blunted SmaI-NheI restriction sites of pBSI, and then the human GFAP promoter (34,35) was inserted into the HindIII-EcoRV restriction sites upstream of the chimeric intron. Founder lines were generated by injecting the 4.1-kilobase SpeI-HindIII fragment of the GFAP-E2F1 transgene into Friend virus B susceptibility hybrid zygotes. Transgenic founders were developed by the Transgenic Animal Facility at the M. D. Anderson Cancer Center, Science Park-Research Division. Transgenic lines were maintained by breeding founders with a Friend virus B wild-type strain. Animal studies were performed in the veterinary facilities at M. D. Anderson Cancer Center in accordance with institutional guidelines.

Isolation of Cortical Astrocytes

Normal mouse astrocytes and transgenic GFAP-E2F1 mouse astrocytes were obtained from the brain cortex of postnatal day 4 mice as previously described (36,37). Cultures were maintained in Dulbecco's modified Eagle medium–F-12 medium (1:1; Gibco) supplemented with 10% fetal bovine serum. To determine the number of cell population doublings, cells were seeded in six-well plates at a density of 104 cells per well and counted after 6 days. The number of population doublings was calculated according to the formula: log(Nh) – log(Ni) = xlog 2, where Nh is the number of cells harvested, Ni is the number of cells inoculated, and x is the number of population doublings (38). The experiment was carried out three times with quadruplicate samples.

Patient Selection

All tumors were glioblastoma multiforme. The two separate groups of fresh-frozen glioblastoma multiforme tissues (WHO grade IV astrocytoma) were obtained from patients enriched for long-term survival (i.e., alive ≥2 years after diagnosis; Table 1). The first group contained 34 samples from the tissue bank at the Brain Tumor Center in the Department of Neurological Surgery, University of California, San Francisco, that were included in an expression array study (U95Av2; Affymetrix, Santa Clara, CA), for which detailed methodology and validation are published elsewhere (39). We studied E2F1 and hTERT mRNA levels in that cohort of patients. Five samples of non-neoplastic brain obtained from patients undergoing surgery for temporal lobe epilepsy were used for comparison. A second group of 27 fresh-frozen glioblastoma multiforme tissues (WHO grade IV astrocytoma) was selected from the tissue bank at the Brain Tumor Center at M. D. Anderson Cancer Center, Houston. E2F1 protein levels were measured in these samples by immunoblotting. All tumors were from patients with newly diagnosed glioblastoma multiforme who had received no therapy before sample collection. All patients underwent gross total resection of their tumors, as assessed by postoperative imaging studies, and had completed conventional external beam radiation therapy after surgery. The median Karnofsky performance score of the 61 patients was 90. The glioblastoma multiforme tumor samples contained at least 90% tumor, as assessed in a section cut from the tumor. The local institutional review boards approved the study, and all patients signed consent for specimens to be used for research purposes. Overall survival was determined as the interval from initial surgery to death and was obtained from the patient's medical record or the Social Security Death Index.


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Table 1.  Clinical profile and molecular analysis of patients with glioblastoma multiforme*

 
Affymetrix Expression Array

E2F1 and hTERT mRNA levels were analyzed from a microarray database containing information on 34 fresh-frozen glioblastoma multiforme tissues (WHO grade IV astrocytoma), as reported previously (39). Briefly, test arrays were used first to determine the quality of the complementary RNA (cRNA), and 15 µg of cRNA was hybridized to U95Av2 human GeneChip expression arrays, according to the manufacturer's specifications (Affymetrix). Scanning and image analysis of the arrays were performed according to the manufacturer's protocols. Data were analyzed by use of the PerfectMatch algorithm, as previously described (40). Expression data for a small proportion of the probe sets were related to the batch in which the samples were processed due to technical differences in processing, and this set of genes (approximately 5% of the total) was excluded from further analysis. Data were also analyzed by use of the Affymetrix Microarray program suite (MAS version 5.0). Similar results were obtained with the two methods. Information on validation of the expression array (by real-time RT–PCR and immunohistochemistry) has been published elsewhere (39).

Statistical Analyses

For in vitro experiments, statistical analyses were performed with two-tailed Student's t tests. Data are expressed as mean and 95% confidence interval (CI). The correlation between E2F1, E2F5, and telomerase expression and activity was assessed with Spearman's rank test. Survival was assessed by the Kaplan–Meier method, and comparisons were analyzed with the log-rank test. To analyze survival data in the second set of sample patients, patients were reclassified into two groups by E2F1 protein expression as determined from immunoblots (low expression <1 or high expression ≥1) according to the martingale residual plots. Univariate analysis and multivariable models were fit by use of a Cox proportional hazards regression model. The proportional hazards assumption was evaluated by graphical displays and determined to be adequate for the purpose of this assessment. All statistical tests were two-sided.


    RESULTS
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 Notes
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
E2F1-Mediated Regulation of hTERT Promoter Activity in Cancer Cells

To investigate the putative role of E2F1 in regulating telomerase activity, we selected two telomerase-positive human cancer cell lines [U-251 MG and HeLa (17)] and one telomerase-negative cell line [Saos-2 (13)] (Fig. 1, A). Adenoviral-mediated overexpression of exogenous E2F1 increased hTERT promoter activity in U-251 MG cells (9.5-fold, 95% CI = 8.2-fold to 10.7-fold; P<.001), in HeLa cells (11.3-fold, 95% CI = 9.8-fold to 12.7-fold; P<.001), and also in telomerase-negative Saos-2 cells (8.7-fold, 95% CI = 7.8-fold to 9.5-fold; P<.001), all compared with basal activity in corresponding mock-infected cells (Fig. 1, B). Overexpression of exogenous E2F1 in Saos-2 cells increased levels of hTERT mRNA and protein (Fig. 1, C and D) and also reactivated telomerase activity (Fig. 1, E), without modifying the mRNA levels of the hTR, the RNA moiety of telomerase. Thus, overexpression of E2F1 results in hTERT transcription, increased hTERT protein expression, and the reactivation of the enzymatic function of hTERT.



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Fig. 1. E2F1 and transcriptional regulation of the expression of hTERT, the catalytic subunit of human telomerase. A) Analysis of basal telomerase activity in a panel of cancer cell lines, including HeLa (positive control), heat-treated (HT) HeLa (negative control), U-251 MG, and Saos-2 cells with the telomere repeat amplification protocol (TRAP). IC = internal control; bp = base pair(s). B) hTERT promoter activity after transfer of E2F1 cDNA to cells. Cells were transfected with an hTERT promoter–luciferase reporter construct and 1 hour later were treated with adenovirus carrying E2F1 (open bars), adenovirus carrying cytomegalovirus (CMV; solid bars), or adenovirus carrying {beta}-galactosidase ({beta}-Gal, hatched bars) constructs or were mock-treated (shaded bars). After 24 hours, cells were harvested, and luciferase activity was measured. All values were normalized to the expression of renilla luciferase; data are expressed as relative to those of mock-infected cells (arbitrary value of 1 U). Data are the means of five experiments. Error bars = 95% confidence intervals. For U-251 MG cells (*), P<.001; for HeLa cells ({bullet}), P<.001; and for Saos-2 cells (+), P<.001. C) Detection of hTERT mRNA expression in Saos-2 cells. Reverse transcriptase–polymerase chain reaction (PCR) was performed 24 or 48 hours after transfer of E2F1 cDNA. Shown are levels of the RNA component of telomerase hTR and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a loading control. D) hTERT protein expression in Saos-2. Immunoblotting was performed 24 hours transfer of E2F1 cDNA, as indicated. The percentage of cells in S phase, indicated below the lanes, was determined in duplicate samples by flow cytometry. Loading control was {beta}-actin. Results are representative of those from three independent experiments, all with similar results. E) Detection of telomerase activity in Saos-2 cultures. TRAP was performed at the indicated time points after infection with an adenovirus carrying E2F1 cDNA. F) Interaction between E2F1 protein and the hTERT promoter. The chromatin immunoprecipitation assay was used to investigate this interaction. Cells were collected 24 hours after infection with an adenovirus carrying E2F1 cDNA and then incubated with antibodies ({alpha}) against E2F1 or immunoglobulin G (IgG). We used a nonspecific mouse IgG as negative control for the immunoprecipitation (IP) and a plasmid containing the hTERT promoter as a positive control [(+) C] for the PCR assay. Ten percent of the total cell lysate before immunoprecipitation was subjected to PCR to determine the chromatin input, as shown at the bottom.

 
Physical Interactions Between Transcription Factor E2F1 and the hTERT Promoter

The hTERT promoter sequence contains at least three putative E2F1 binding sites in the proximal region of the promoter (21,41). To determine whether E2F1 can bind to the hTERT promoter, we transferred exogenous E2F1 into Saos-2 cells and examined the interaction between E2F1 and the hTERT promoter with a ChIP assay. We detected an interaction between E2F1 protein and the hTERT promoter within 24 hours of transferring exogenous E2F1 (Fig. 1, F). As expected, we detected low levels of interaction between the hTERT promoter and E2F1 in control Ad-CMV–infected or mock-infected cells.

E2F1-Mediated Induction of Telomerase Activity in Saos-2 Cells

To ascertain whether endogenous E2F1 levels were associated with the induction of functional telomerase, we determined the S-phase fraction in Saos-2 cultures synchronized by serum starvation and measured telomerase activity by the TRAP assay in these cultures. At 36 and 48 hours after serum-starvation release, S-phase cells represented 45.5% (95% CI = 36.8% to 54.1%) and 53.8% (95% CI = 47.3% to 60.2%) of cell populations in the cultures, respectively (P<.001), compared with that in cells at 0 hour (Fig. 2, A). In addition, compared with cultures at 0 hour, corresponding S-phase–enriched cultures contained increased levels of E2F1 protein and increased telomerase activity that increased progressively with time (Fig. 2, B).



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Fig. 2. Telomerase activity in synchronized Saos-2 cultures. A) Progressive accumulation of S-phase cells in synchronized Saos-2 cell cultures. The percentage of cells in S-phase was determined by the DNA content of cells as assessed by flow cytometry. Percentages of Saos-2 cells in the S phase of the cell cycle after serum-starvation release are shown below the histograms as the mean of three independent experiments and its corresponding 95% confidence intervals. Arrowheads indicate the peaks for the 2n (G0/G1 phase) and 4n (G2/M phase) cell populations. B) Telomerase activity as assessed by the telomere repeat amplification protocol (TRAP) after cell cycle block release in HeLa (positive control), heat-treated (HT) HeLa (negative control), and Saos-2 cells. Activity of hTERT, the catalytic subunit of human telomerase, was assessed at 0, 12, 24, 36, and 48 hours after releasing Saos-2 cells from serum starvation; it was detected at 36 hours and had increased at 48 hours. E2F1 protein was detected by immunoblot analysis at 24 hours after serum block release, and its level had increased at 36 hours and at 48 hours. Saos-2 cells were collected at the indicated times after serum stimulation and analyzed by immunoblotting. {beta}-Actin was the loading control for the immunoblot. C) Detection of hTERT expression in Saos-2 cells. Reverse transcriptase–polymerase chain reaction to detect hTERT mRNA (upper panel) and immunoblot analysis to detect hTERT protein (lower panel) were performed at the indicated points after wild-type (WT) adenoviral infection. Glyceraldehyde-3-phosphate dehydrogenase (GADPH) was used as the loading control for PCR, and {beta}-actin was the loading control for immunoblotting. Expression of adenoviral E1A protein and increased expression of E2F1 protein are observed. The percentage of cells in the S phase of the cell cycle is indicated below the immunoblot (values were representative of three independent experiments). UVi = UV-inactivated WT adenovirus; mock = mock-infected. D) Detection of telomerase activity in Saos-2 cultures. TRAP was performed at the indicated times after infection with WT adenovirus or UV-inactivated WT adenovirus or mock infection. HeLa was the positive control, and HT HeLa was the negative control. IC = internal control.

 
To confirm whether the endogenous E2F1 participated in the induction of hTERT expression, we examined the effect of the Rb-binding adenoviral oncoprotein E1A on the expression of hTERT mRNA and protein. We expected that the E1A-mediated release of E2F1 transcriptional activity would result in increased levels of telomerase. In fact, we found that infection of Saos-2 cells with Ad-WT increased both E2F1 transcriptional activity and hTERT promoter activity (11-fold increase, 95% CI = 10.5-fold to 11.2-fold; P<.001), compared with those in mock-infected cells (data no shown). Adenoviral-mediated induction of endogenous E2F1 was also associated with detectable levels of hTERT mRNA and protein (Fig. 2, C) and reactivation of telomerase activity in Saos-2 cultures (Fig. 2, D). Thus, increased expression of endogenous E2F1 appears to be associated with increased hTERT transcription and increased telomerase activity.

Restoration of the Rb Pathway and hTERT Expression

We next investigated whether restoring the Rb pathway in telomerase-positive U-251 MG cells would reduce the transcriptional activity of E2F1 and thus reduce hTERT expression and telomerase activity. Within 24 hours of transfer, Rb or p21 cDNAs arrested U-251 MG cells in G0–G1 phase of the cell cycle (G0–G1/S-phase cell ratio in Ad-Rb–treated cultures = 7.6, 95% CI = 6.1 to 9, P<.001; in Ad-p21–treated cultures = 13.0, 95% CI = 11.6 to 14.3, P<.001; and in Ad-CMV–infected cultures = 3.5, 95% CI = 2.5 to 4.4). Treating these cells with the cyclin-dependent inhibitor olomoucine (42) also statistically significantly increased the ratio of G0–G1/S-phase cells by 48 hours after treatment (9.5, 95% CI = 8.4 to 10.5) compared with that in mock-treated cultures (3.8, 95% CI = 2.4 to 5.1) (difference = 5.7, 95% CI = 3.9 to 7.7; P<.001). We detected a statistically significant reduction of hTERT promoter activity 24 hours after treatment. The reduction in hTERT promoter activity was statistically significant and strongest in cells treated with Ad-Rb (20% of control luciferase activity, 95% CI = 18.8% to 21.1%; P<.001) compared with that in cells treated with Ad-CMV. Transfer of p21 cDNA (65%, 95% CI = 62.7% to 67.2%; P<.001) or treatment with olomoucine (48%, 95% CI = 46.7% to 49.2%; P<.001) also resulted in moderate and statistically significant inhibition of hTERT promoter activity, compared with that in Ad-CMV–treated cell cultures (Fig. 3, A). Expression of hTERT mRNA could not be detected after transferring pRb or p21 cDNAs or treatment with olomoucine (Fig. 3, B). The reductions in hTERT promoter activity and mRNA expression were also associated with reductions in the hTERT protein levels (Fig. 3, C). Finally in U-251 MG cells, when the Rb pathway was restored, telomerase activity could not be detected (Fig. 3, D).



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Fig. 3. Restoration of the retinoblastoma (Rb) pathway and expression of hTERT, the catalytic subunit of human telomerase. A) hTERT promoter activity assessed by a luciferase reporter assay (as described in Fig. 1, B). hTERT promoter activity was assayed in U-251 MG cells 24 hours after the transfer of pRb or p21 cDNA or treatment with 1 µM olomoucine (Olo). All values were normalized to the expression of renilla luciferase; data are expressed relative to those of mock-treated cells (mock; arbitrary value of 1 U). Cultures infected with Ad-{beta}-Gal ({beta}-gal) were used as control for specificity. *P<.001, compared with cells infected with adenovirus carrying cytomegalovirus (CMV). Data are the mean of five independent experiments. Error bars = 95% confidence intervals. B and C) Modulation of the Rb-E2F1 pathway and the expression of hTERT mRNA (B) and protein (C) in U-251 MG cells. hTERT mRNA was assessed by reverse transcriptase–polymerase chain reaction, and hTERT protein was assessed by immunoblotting at the indicated times after mock infection; infection with adenovirus carrying CMV, pRb cDNA, or p21 cDNA; or treatment with 1 µM olomoucine (Olo). Glyceraldehyde-3-phosphate dehydrogenase (GADPH) was the loading control. D) Telomerase activity after the transfer of pRb or p21 cDNAs or treatment with 1 µM olomoucine in U-251 MG cells. Telomerase activity was assessed by the telomere repeat amplification protocol in lysates of U-251 MG cells at the indicated times after treatment. Histone 3 was the loading control. IC = internal control.

 
E2F1-Mediated Regulation of Telomerase in Normal Murine Glial Cells

Telomerase activity in the mouse brain decreases shortly after birth (43). To investigate the role of E2F1 in the regulation of telomerase in glial cells, we isolated astrocytes from E2F1 transgenic mice and wild-type mice. E2F1 protein levels were higher in astrocytes derived from E2F1 transgenic mice than in those derived from their counterpart wild-type mice (i.e., normal mouse astrocytes) (Fig. 4, A). The modification in E2F1 expression was related to the proliferative phenotype of the cultures. That is, during a 6-day period, the population size of normal mouse astrocytes doubled three times (95% CI = 1.4 to 4.5 times) and that of transgenic E2F1 astrocytes doubled five times (95% CI = 3.5 to 6.4 times).



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Fig. 4. Telomerase activity in transgenic E2F1 astrocytes. A) Expression of mTERT, the catalytic subunit of mouse telomerase, and E2F1 protein in GFAP-E2F1 astrocytes from transgenic E2F1 (tgE2F1) mice and normal mouse astrocytes (NMAs). Proteins were assessed by immunoblot analyses, with {beta}-actin as the loading control. B) hTERT promoter activity was assayed by use of hTERT-driven luciferase activity in NMA and tgE2F1 astrocytes. Higher basal hTERT promoter activity was detected in tgE2F1 astrocytes than in NMAs (P<.001). Transferring E2F1 (*P<.001) or retinoblastoma (Rb) ({bullet}, P = .005) cDNAs modulated this activity, relative to that in mock-infected cells. Data are the mean of five independent experiments. Error bars = 95% confidence intervals. CMV = cytomegalovirus. C) mTERT mRNA in astrocytes and the transfer of Rb cDNA. mRNA was assessed by reverse transcriptase–polymerase chain reaction(PCR) in normal and transgenic astrocytes and 24 hours after the transfer of E2F1 or Rb cDNAs. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the loading control. D) Telomerase activity in astrocytes from normal and tgE2F1 mice and after the transfer of E2F1 and Rb cDNAs and the percentage of cells in S phase. The telomere repeat amplification protocol was used to measure telomerase activity. The DNA content of cells as assessed by flow cytometry was used to determine the percentage of cells in S phase. HeLa was the positive control, and heat-treated (HT) HeLa was the negative control. IC = internal control. Percentages of cells in S phase in parallel astrocyte cultures are shown below the lanes. E) Chromatin immunoprecipitation (ChIP) assay and the interaction between E2F1 protein and the mTERT promoter in NMA and tgE2F1 astrocytes. Putative E2F-responsive elements are shown by boxes that contain sequences of primers used; predicted PCR product sizes in base pairs (bp) are indicated as A, B, and C. Cultures were collected and incubated with antibodies ({alpha}) against E2F1 or immunoglobulin G (IgG). The negative control [(–) C] for the immunoprecipitation (IP) was a nonspecific mouse IgG. The positive control [(+) C] for the PCR assay was a plasmid containing the mTERT promoter. Ten percent of the total cell lysates was subjected to PCR before IP to determine the chromatin input, as shown at the bottom of the panel.

 
Transgenic E2F1 astrocytes contained increased levels of mTERT protein (Fig. 4, A) and of promoter activity (7.0-fold increase, 95% CI = 6.5-fold to 7.4-fold; P<.001), compared with the low level of basal transcriptional activity observed in normal mouse astrocytes (Fig. 4, B). Moreover, expression of exogenous E2F1 in normal mouse astrocytes resulted in increased hTERT promoter activity (8.5-fold, 95% CI = 7.8-fold to 9.1-fold; P<.001), compared with mock-, Ad-CMV–, or Ad-{beta}-gal–treated normal mouse astrocytes. Furthermore, transfer of Rb cDNA to transgenic E2F1 astrocytes reduced hTERT transcriptional activity (P = .005), compared with the level observed in mock- or Ad-CMV–treated transgenic E2F1 astrocytes, to a level similar to the level observed in normal mouse astrocytes (Fig. 4, B). In agreement with these data, the mTERT transcript was present in transgenic E2F1 but was dramatically reduced in transgenic E2F1 astrocytes transfected with pRb cDNA (Fig. 4, C). Finally, we observed telomerase activity in astrocytes from postnatal day 4 transgenic E2F1 mice but not in control normal mouse astrocytes (41). However, telomerase activity was clearly noticeable in normal mouse astrocytes expressing ectopic E2F1. Transfer of pRb to transgenicE2F1 astrocytes reduced mTERT expression and telomerase activity (Fig. 4, D).

Analyses of the mTERT promoter sequence identified four possible binding sites for E2F1 (Fig. 4, E). Experiments with different combinations of primers showed that the putative E2F-binding site was located between positions –1682 and –1313 of the mTERT promoter. This direct interaction between E2F1 and the mTERT promoter was detected in astrocytes isolated from wild-type and E2F-transgenic animals, suggesting that E2F1 directly interacts with the mTERT promoter in normal mouse glial cells (Fig. 4, E).

Correlation of E2F1 and hTERT Expression Levels in Glioblastoma

We next examined the mRNA expression levels of E2F1 and hTERT in a series of human glioblastoma multiforme specimens (Table 1). We found a correlation between E2F1 mRNA expression levels and hTERT mRNA expression levels (R = .8; P<.001) (Fig. 5, A). To demonstrate the specificity of this correlation, we found that hTERT mRNA expression was not correlated (R = –.04) with the expression of E2F5, another E2F family member whose activity is not involved in the positive transcriptional activation of cell cycle progression. Thus, E2F1 may be involved in triggering or maintaining hTERT expression in malignant gliomas and, therefore, favoring the perpetuation of the neoplastic phenotype in these tumors.



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Fig. 5. Expression of E2F1 and hTERT, the catalytic subunit of human telomerase, in human glioblastoma multiforme (GBM). A) E2F1 and hTERT mRNA expression in a set of 34 human GBM samples. Scatter plot and loess smoothed line illustrate the correlation between hTERT and E2F1 expression levels. B) Kaplan–Meier analysis of the association between survival and the expression of hTERT and E2F1 mRNA. The differences in overall survival were statistically significant among the four groups (log-rank test, P = .015). The 4-year overall survival rates were 20% (95% confidence interval [CI] = 7 to 55) for the low-E2F1/low-hTERT group, 50% (95% CI = 13 to 100) for the low-E2F1/high-hTERT group, and 7% (95% CI = 1 to 44) for the high-E2F1/high-hTERT group. C and D) Kaplan–Meier survival curves stratified by hTERT (C) or E2F1 (D) mRNA levels, with prognostic significance by the log-rank test using the median values as cut points. The probability of survival (%) and corresponding 95% confidence levels are shown below the graphs. E) E2F1 protein expression in two GBM samples (GBM A and GBM B = low and high E2F1 expression, respectively) from the second set of 27 GBM samples. Representative immunoblots are shown; E2F1 expression level is expressed as relative to the expression level of tubulin. Controls were lysates from U-251 MG cells infected with adenovirus carrying an E2F1 cDNA [(+) C] and from normal human astrocytes (NHA). Patient survival data are shown at the bottom. F) Kaplan–Meier survival curves stratified by E2F1 protein expression levels in the second set of 27 GBM samples, with prognostic significance by the two-sided log-rank test. All statistical tests were two-sided.

 
E2F1 and hTERT as Prognostic Factors in Malignant Gliomas

Because we observed a strong correlation between hTERT expression and E2F1 expression in glioblastoma multiforme samples and because hTERT expression has been associated with reduced survival in glioma patients (44,45), we investigated whether E2F1 and hTERT mRNA levels in the glioblastoma multiforme samples were associated with patient survival. We used the median values for E2F1 expression (i.e., 234.1 arbitrary fluorescent units) and hTERT expression (i.e., 347.61 arbitrary fluorescent units) as cut points to divide patients into the following four groups: those with low expression of E2F1 and low expression of hTERT (n = 15); those with high expression of E2F1 and low expression of hTERT (n = 2); those with low expression of E2F1 and high expression of hTERT (n = 2); and those with high expression of E2F1 and high expression of hTERT (n = 15). The differences in overall survival were statistically significant among the four groups (log-rank test, P = .015) (Fig. 5, B). We evaluated potential differences among these four groups by age and sex with the adjusted Cox proportional hazards regression model. The analyses showed that high expression of E2F1 plus high or low expression of hTERT was negatively associated with survival (P = .009 and P = .05, respectively), compared with low expression of both genes. However, by conditioning on low-expressing E2F1 tumors, hTERT expression levels were not statistically significantly associated with patient survival (P = .93).

Low hTERT mRNA expression in tumors was associated with longer median survival than high hTERT mRNA expression in tumors. The median overall survival was 97.9 weeks (95% CI = 60.9 to 206 weeks) for patients with low tumor hTERT expression compared with 46.1 weeks (95% CI = 31.1 to 83 weeks) for patients with high tumor hTERT expression (difference = 51.8 weeks, 95% CI = 2.5 to 146; log-rank test, P = .038) (Fig. 5, C). In addition, low E2F1 mRNA expression in tumors was associated with a longer median survival (103.6 weeks, 95% CI = 82 to 227) than was high E2F1 mRNA expression in tumors (46.1 weeks, 95% CI = 31.1 to 80) (difference = 57.5 weeks, 95% CI = 14.7 to 159.7; log-rank test, P = .002) (Fig. 5, D). The one study subject who was still alive at the end of this study was a member of the group of patients with tumors with low E2F1 and low hTERT expression.

Next, we assessed whether the association between overall survival and the expression levels of E2F1 and hTERT was modified by age (44.5 years, 95% CI = 44.2 to 47.7 years) and sex (male to female ratio = 1.13/1). The results were consistent with the log-rank analysis; i.e., the expression of E2F1 and hTERT was statistically significantly associated with overall survival of patients with glioblastoma multiforme (P = .003 and P = .042, respectively), but age and sex were not associated with overall survival (P = .38 and P = .71, respectively).

Prognostic Value of E2F1 in Human Glioblastomas

Transcription factor E2F1 regulates the expression of genes involved in many cell functions in addition to telomerase (46). To determine whether overexpression of E2F1 mRNA could be used as a prognostic factor that was independent of telomerase expression, we investigated whether E2F1 protein expression was associated with overall survival, by use of an adjusted Cox proportional-hazards regression model by age and sex. We found that E2F1 protein expression was the only factor that statistically significantly affected overall survival (P = .04). Although hTERT was statistically significantly associated with survival in the univariate Cox proportional hazards regression analysis, the adjusted analysis for age and sex showed that, in the presence of E2F1 protein expression, hTERT protein expression was not statistically significantly associated with survival (P = .62). Furthermore, tumors with a high level of E2F1 mRNA expression were associated with a 3.94-fold higher risk of death (95% CI = 1.07-fold to 14.52-fold) than that associated with tumors with a low level of E2F1 mRNA expression.

Although protein extracts from tumors were not available from the same sample cohort that was examined by the expression array methodology, we used specimens from an independent group of 27 glioblastoma multiforme patients to examine the expression of E2F1 protein (Table 1). We found that E2F1 protein levels were higher in protein extracts from these tumor specimens than that in protein extracts from normal human astrocytes (Fig. 5, E), which is consistent with the higher levels of E2F1 mRNA observed in the expression array of glioblastoma multiforme compared with that in normal brain (P = .018, Wilcoxon rank sum test). Moreover, the level of E2F1 protein expression was statistically significantly associated with overall survival (P<.001, log-rank test) (Fig. 5, F). In addition, consistent with the expression array data, low E2F1 protein expression in tumors was associated with longer survival (median survival time = 220 weeks, 95% CI = 126 to [not available]) than was high E2F1 protein expression in tumors (22.7 weeks, 95% CI = 11.6 to [not available]). Results from the univariate Cox proportional hazards regression model analysis were consistent with the log-rank analysis, showing that the expression of E2F1 protein was associated with overall survival of patients with glioblastoma multiforme (P<.001). Although age (57.2 years, 95% CI = 51.4 to 62.97) was also associated with overall survival (P = .011) in the multivariable analysis, only E2F1 protein expression was associated with the survival rate, increasing the risk of death 25.7-fold (95% CI = 4.97-fold to 133.81-fold).


    DISCUSSION
 Top
 Notes
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
We found that the Rb-E2F pathway was involved in the regulation of telomerase expression and activity in cancer and normal cells, and we also found a statistically significant correlation between the levels of E2F1 and hTERT mRNA and protein in tissue samples of malignant brain tumors. Specifically, hTERT mRNA and protein levels, as well as telomerase activity, increased in cells overexpressing E2F1 protein. Moreover, the increased expression of hTERT mRNA and protein in cancer cells was suppressed by restoration of Rb pathway function. Isogenic astrocytes that differed only in their levels of E2F1expression had markedly different levels of hTERT expression and activity, suggesting that the expression of E2F1 protein in normal cells may be sufficient to trigger telomerase activity. Moreover, E2F1 and hTERT levels were strongly associated with survival in patients with glioblastoma multiforme, with E2F1 being the more strongly associated factor in multivariable analysis.

Our finding that E2F1 directly binds the hTERT promoter provides a mechanistic link that underlies the regulation of telomerase activity during cell cycle progression. In this model, free E2F1 activates telomerase in normal and cancer cells, and the Rb-E2F1 complex functions by repressing telomerase expression and activity. Our data support previous observations (4749) about the role of Rb protein in repressing telomerase activity, and Rb protein may be at least partially responsible for the block of cell cycle progression and the decreased telomerase activity that is observed in quiescent cells cultured in the absence of serum (50,51). Furthermore, in normal cells, Rb protein is part of the E2F complexes that bind the hTERT promoter during G0/G1 phases, but not during S phase (23). Intriguingly, Rb and Rb-related proteins directly regulate telomere length, and they do so independently of the regulation of telomerase activity and the roles of Rb protein in the cell cycle (52). Fibroblasts that do not express the Rb pocket proteins (i.e., pRb, p107, and p130) have the ability to rapidly elongate their telomeres, bolstering the possibility that Rb family members have a direct role in regulating telomere length or telomere structure. Thus, multiple complex relationships between Rb protein and telomerase activity appear not only to govern telomerase function during the cell cycle but also to control chromosome structure and genomic stability beyond the strict limits of cell cycle regulation. The Rb pathway may also be involved in regulating hTR, the mRNA component of the telomerase complex (53,54).

In normal cells evading Rb-mediated regulation of cell cycle progression, the resultant free E2F1 protein can induce hTERT promoter activity (21). We found that mouse astrocytes engineered to constitutively express E2F1 had increased mTERT promoter transcriptional activity, mTERT protein levels, and telomerase activity. In addition, telomerase activity was not detected in tumor tissue from E2F-1–/– mice but was detected in tissue from wild-type mice (55). Thus, the combined results from transgenic and E2F1-null mouse models support the theory that E2F1 has a physiologic role in the regulation of telomerase.

The statistically significant correlation between E2F1 and hTERT mRNA expression in a subset of patients with glioblastoma multiforme confers clinical relevance to the mechanistic association between the expression of E2F1 and hTERT. Lower levels of E2F1 or hTERT mRNA expression were associated with longer median survival in patients with glioblastomas, compared with a higher level of either. Previous reports showed that telomerase activity in tumor cells is associated with progression of malignant gliomas (56), that hTERT expression is associated with reduced survival in patients with malignant gliomas (45), and that the absence of telomerase activity and the presence of the mechanism of alternative lengthening of telomeres are associated with longer survival in patients with glioblastoma multiforme (57).

E2F1 was the factor most strongly associated with the overall survival of patients with high-grade gliomas. A previous study (44) reported that the expression of E2F1 protein was not correlated with the survival of patients with gliomas. Chakravarti et al. (44) also found underexpression of E2F1 in some glioblastoma multiforme tumors. However, the expression of E2F1 in our glioblastoma multiforme samples was consistently the same or higher than that in our control samples (i.e., normal brain tissue and normal human astrocytes). Our results were confirmed at protein and RNA levels in two independent sample cohorts, and a high level of E2F1 expression in both analyses was associated with poor prognosis for patients with glioblastoma multiforme. The discrepancy between these two studies can, at least in part, be explained by differences in the sample cohorts, because we analyzed a cohort enriched for patients with long-term survival.

Although our results are encouraging, our study had some limitations. First, because it made use of archival material, it had the potential biases associated with a retrospective study. In this regard, a prospective study may offer the possibility for yielding definitive conclusions. Moreover, because we were examining two cohorts of patients who were relatively homogeneous in terms of prolonged survival, we cannot rule out the possibility that our results would be muted in a more heterogeneous group of patients. In addition, it would be premature to consider E2F1 expression as a reliable prognostic marker for clinical use, because more standard tests, such as immunohistochemistry or quantitative PCR, must be done to validate our conclusions. In addition, our conclusions pertain only to glioblastomas, and studies in other types of solid tumors are required to broaden the role of E2F1 in the telomerase biology of these other tumors.

In conclusion, we have presented evidence that the level of E2F1 expression is strongly associated with survival of glioblastoma patients. This association deserves further analysis in prospective studies. Because the treatment of glioblastoma multiforme is multimodal and involves several levels of intervention, the identification of molecular markers that are active early in the development of this disease, such as E2F1 expression and telomerase activity, should facilitate the screening and selection of those patients who require an intense treatment regimen as soon as the disease is diagnosed.


    NOTES
 Top
 Notes
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Supported by grant RO1-CA80748 from the National Cancer Institute.

We thank Joann Aaron, Department of Neuro-Oncology, University of Texas M.D. Anderson Cancer Center, Houston, TX, and Vicky J. Williams, Department of Scientific Publication, University of Texas M.D. Anderson Cancer Center, Houston, TX, for editorial assistance.


    REFERENCES
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 

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Manuscript received February 22, 2005; revised August 24, 2005; accepted September 9, 2005.



             
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