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).
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ABSTRACT |
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INTRODUCTION |
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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.
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PATIENTS AND METHODS |
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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--gal (Ad-
-gal, where
-gal is
-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 promoterluciferase 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--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 promoterdriven 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 (20100 µg, depending on the antibody used) were subjected to sodium dodecyl sulfateTris-glycine gel electrophoresis and transferred to nitrocellulose membranes, as described elsewhere (28). Blots were incubated with the antibodies against E2F1 (product KH-95) and -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 peroxidaseconjugated 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 TranscriptasePolymerase 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 mediumF-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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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 RTPCR 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 KaplanMeier 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.
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RESULTS |
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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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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-CMVinfected 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-phaseenriched cultures contained increased levels of E2F1 protein and increased telomerase activity that increased progressively with time (Fig. 2, B).
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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 G0G1 phase of the cell cycle (G0G1/S-phase cell ratio in Ad-Rbtreated cultures = 7.6, 95% CI = 6.1 to 9, P<.001; in Ad-p21treated cultures = 13.0, 95% CI = 11.6 to 14.3, P<.001; and in Ad-CMVinfected 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 G0G1/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-CMVtreated 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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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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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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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).
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DISCUSSION |
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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.
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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.
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Manuscript received February 22, 2005; revised August 24, 2005; accepted September 9, 2005.
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