Differential transcriptional regulation of human telomerase in a cellular model representing important genetic alterations in esophageal squamous carcinogenesis
Michael Quante,
Steffen Heeg,
Alexander von Werder,
Gitta Goessel,
Christine Fulda,
Michaela Doebele,
Hiroshi Nakagawa 1,
Roderick Beijersbergen 2,
Hubert E. Blum and
Oliver G. Opitz *
Department of Medicine and Institute of Molecular Medicine and Cell Research, University of Freiburg, Freiburg, Germany, 1 Gastroenterology Division, University of Pennsylvania, Philadelphia, PA, USA and 2 Netherlands Cancer Institute, Amsterdam, The Netherlands
* To whom correspondence should be addressed at: Department of Medicine II and Institute of Molecular Medicine and Cell Research, University of Freiburg, Hugstetter Strasse 55, 79106 Freiburg, Germany. Email: opitz{at}med1.ukl.uni-freiburg.de
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Abstract
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Telomerase activity is observed in
90% of human cancer including esophageal squamous cell cancer. Normal somatic cells do not display telomerase activity on a regular basis. The major mechanism to regulate telomerase activity in human cells is the transcriptional control of the catalytic subunit, the human reverse transcriptase gene hTERT. However, the manner in which telomerase activity is regulated during malignant transformation and whether this regulation is influenced by single genetic alterations important in this process are not well understood. In this study we investigated the transcriptional regulation and activity of human telomerase in a cellular model representing important known genetic alterations observed in esophageal cancer. We characterized the respective cells with regard to their telomere biology and telomerase expression, transcriptional regulation using promoter- as well as electrophoretic mobility shift assay-analyses and their promoter methylation status. We could demonstrate that telomerase expression and subsequent activity are differentially regulated in the progression from normal esophageal epithelial cells to genetically defined esophageal cells harboring a specific genetic alteration frequently found in esophageal cancer and compared those changes with esophageal cancer cells. Whereas primary esophageal cells are mainly regulated by Sp1, in cells harboring a genetic alteration as cyclin D1 overexpression other transcription factors like E2F and c-myc as well as promoter methylation influence hTERT transcription. This model demonstrates that the transcriptional regulation of telomerase is influenced by a given genetic alteration important in esophageal cancer, and therefore provides new insight in telomerase regulation during carcinogenesis.
Abbreviations: EMSA, electrophoretic mobility shift assay; MSP, methylation-specific PCR; TRAP, telomeric repeat amplification protocol
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Introduction
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The process of malignant transformation from normal human somatic cells to immortal tumor cells involves complex regulatory mechanisms. This process is characterized by the acquisition of mutant alleles of oncogenes and/or tumor suppressor genes and other genetic changes that directly or indirectly control cell proliferation and malignant transformation (1,2). In addition to changes in oncogenes and tumor suppressor genes, the maintenance of telomeres is an important regulator of immortalization and transformation (35). Telomeres are protective sequences that constitute the end of chromosomes with a thousand double-stranded repeats of the sequence TTAGGG. Each cell division is associated with the loss of 30150 bp of telomeric DNA and critically short telomeres eventually induce normal cells to senesce.
Normal or primary esophageal epithelial cells (EPCs) display a restricted replicative live span in cell culture eventually entering a state of permanent growth arrest, called replicative senescence (6). Tumor cells as well as immortalized and transformed cells escape this growth control checkpoint and, therefore, can provide some insight into regulatory mechanisms involved in the processes of immortalization and senescence.
Esophageal squamous cancer cells harbor some of the best studied genetic alterations in cancer development. Cyclin D1 overexpression is one of the most common known genetic alterations in esophageal squamous cell cancer (7) and induces esophageal dysplasia in transgenic mice (8). Cyclin D1 overexpression is considered an early event in esophageal carcinogenesis. Tumor suppressor gene inactivations, such as p53 mutations and p16INK4a deletion, mutation or hypermethylation are also frequently found in human esophageal squamous cell cancer (9,10). All these genetic alterations have been shown to play a critical role in the process of cellular immortalization (2). Immortalization is thereby an important step in the process of the malignant transformation of human cells.
In
90% of all human tumors, including esophageal squamous cell carcinoma, maintenance of telomeres and thus replicative immortality is achieved through uncontrolled activation of telomerase. In contrast, normal human somatic cells appear to have a cell-cycle-dependent activation of telomerase (11). The functional telomerase complex consists of multiple protein components, a structural RNA component that contains the template region binding to the TTAGGG repeats, and most importantly, the human telomerase reverse transcriptase (hTERT) (12,13). Introduction of the hTERT gene in normal cells is sufficient to immortalize most cells and to bypass senescence through the induction of telomerase activity (6,14). hTERT itself appears to be regulated through changes in the rate of transcription, since there is a striking correlation between hTERT mRNA and telomerase activity (15) as well as transcriptional activity in reporter gene assays (16). Although post-transcriptional regulation of hTERT expression through alternative splicing has been observed (17,18) and various post-translational modifications have been discussed (19,20), transcriptional control of the gene seems to be the predominant rate limiting step of hTERT expression (21).
The transcriptional regulation of hTERT expression, has been extensively analyzed in cancer cells (16,2224). Nevertheless, the regulatory mechanisms in normal or premalignant cells are not that well understood. The hTERT-promoter is a GC-rich, TATA-less promoter with a 200300 bp core promoter region (23,25). Studies in recent years have identified canonical and non-canonical motifs of numerous transcription factors. Several known transcription factors, including some oncogene and tumor suppressor gene products, are able to influence hTERT transcription. Their binding sites are located within the core promoter region and their role in the basal transcriptional activity of hTERT has been established (2628). One illustration is the c-myc oncoprotein, which binds to two different E-boxes through heterodimer formation with Max proteins, and the c-myc/Max complex activates directly transcription of hTERT. Switching from c-myc/Max binding to Mad/Max can function as a repressor of hTERT promoter activity (2931).
Nevertheless, little is known about the differential regulation of human telomerase in normal and premalignant, immortalized cells harboring a genetic alteration frequently observed in the corresponding tumor compared with cancer cells. Thus, we investigated the transcriptional regulation of hTERT in genetically defined esophageal epithelial cells corresponding to discrete genetic events observed during in vivo tumor development. In this study we used five different cell types: normal esophageal epithelial cells (EPC), genetically defined derivates of EPC, generated to either overexpress dominant-negative p53 (EPC-
p53), cyclin D1 (EPC-D1) or hTERT (EPC-hTERT), respectively, as well as the esophageal squamous cancer cell line TE-12. Whereas in primary esophageal epithelial cells as well as in mortal EPC-
p53 cells, telomerase transcription was regulated by Sp1, immortal, cyclin D1 overexpressing cells involved c-myc and E2F in their transcriptional regulation of hTERT. Immortal EPC-hTERT cells involved mainly E2F in addition to Sp1, whereas in esophageal cancer cells hTERT transcription was mainly regulated by c-myc. Genetically altered cells seemed to be additionally regulated by hypermethylation of the hTERT-promoter. We demonstrate for the first time that the transcriptional activation of human telomerase is differentially regulated in normal cells versus premalignant, mortal and immortal cells versus cancer cells, and that this regulation depends on the specific genetic alteration involved.
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Materials and methods
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DNA plasmids and hTERT-promoter constructs
The hTERT-promoter reporter gene constructs were generated using a 1242 bp hTERT-promoter plasmid designated as full length in this study. Sense primers were designed with a NheI restriction site and antisense primer with a NcoI restriction site (Table I). Deletion constructs were generated by PCR, accounting for potential transcription factor binding sites (2124). The NheI- and NcoI-digested PCR products were agarose gel-purified and ligated into the promoterless luciferase reporter gene construct, pGL3 (Promega) positioning the translation start site (ATG) at +58, which is identical to the luciferase translation start. The retroviral expression vectors pBPSTR-D1 (obtained from S.A.Reeves, MGH Cancer Center, Boston, MA), pBabe-
p53-puro, and pBabe-hTERT-hygro (provided by R.A.Weinberg, Whitehead Institute, Cambridge, MA) have been described previously (3234). The pGL3-promoter plasmid (containing the SV40-promoter, Promega) as well as the GFP-expression vector pLEGFP-N1 (Becton Dickinson) were used to control for transfection efficiency. All plasmids were transformed in DH5
-cells, purified by a modified alkaline lysis method (Qiagen) and verified by DNA sequencing.
Cell lines and retroviral transduction
Primary normal esophageal epithelial cells, designated EPC, were established from normal human esophagus from a surgical specimen (34). EPC and their derivates (EPC-
p53, EPC-D1 and EPC-hTERT) were grown in keratinocyte-SFM (KSFM; Invitrogen) supplemented with 40 µg/ml bovine pituitary extract (Invitrogen), 1.0 ng/ml EGF (Invitrogen), 100 U/ml penicillin, and 100 µg/ml streptomycin (Sigma). The esophageal squamous carcinoma cell line TE-12 was cultured under standard conditions with DMEM (Sigma) supplemented with 10% fetal bovine serum (Sigma), 100 U/ml penicillin, 100 µg/ml streptomycin and L-glutamine (Sigma).
The amphotropic packaging cell line Phoenix A was grown in supplemented DMEM (Sigma) and then transiently transfected with the respective retroviral vector to generate amphotropic retroviruses (33). Retroviral supernatant was harvested 48 h after transfection and filtered through a 0.45 µm filter. Fresh retroviral supernatant was used for infection of exponentially growing EPC cells. Medium containing puromycin (1 µg/ml) for Cyclin-D1 (pBPSTR-D1), or
p53 (pBabe-
p53-puro) or hygromycin (100 µg/ml) for hTERT (pBabe-hTERT-hygro), respectively, was exchanged to start selection 48 h after infection. In each set of experiments, multiple clones were pooled for further processing.
Transient transfections
Transient transfections of all hTERT-promoter constructs were carried out using an improved lipofectamine method (Effectene®, Qiagen). TE-12, EPC-D1 and EPC-hTERT were plated at a density of 1 x 105 cells/6-well, EPC and EPC-
p53 at a density of 2.5 x 104/24-well and transfected 24 h later with 1 or 0.5 µg of the respective luciferase reporter plasmid, respectively. The pGL3 plasmid containing the SV40-promoter and the empty pGL3 plasmid served as positive and negative control, respectively. Thereby, the SV40-promoter activity in a given transfection experiment can be used to control for transfection efficiencies and uniformity. Additionally a GFP expressing vector (pLEGFP-N1) was co-transfected to directly monitor and control for transfection efficiencies. Cells were harvested 48 h after transfection. One hundred microliters of lysate with uniform protein concentrations was measured for 30 s with a Monolight luminometer (Analytical Luminescence Laboratory). Each experiment was performed in triplicate and at least three sets of independent transfection experiments were performed. Values were then expressed as x-fold increase or decrease compared with the full-length promoter. Activities were expressed as the mean of at least three independent transfection experiments.
TRAP assay/telomeric-length assay
Cellular extracts of all five cell types were assayed for telomerase activity using the PCR-based telomeric repeat amplification protocol (TRAP) assay (35). Cellular extracts (100 ng), along with a heat-inactivated control, were used for TRAP assays. Telomere length was measured by hybridizing
-32P-labeled telomeric (CCCTAA) probe to 10 µg of HinfI- and RsaI-digested genomic DNA, as described previously (35).
RTPCR
mRNA of all five cell types was isolated with the RNeasy protocol (Qiagen) from 1 x 106/35 mm-dish cultured cells. The oligonucleotide primers hTRTF3 and hTRTR7 were used to amplify the N-terminal region of the telomerase gene. Total RNA was extracted as described previously (36) and reverse transcribed using the SuperscriptTM RNase HReverse Transcriptase Systems (Invitrogen). Following a single cycle of reverse transcription at 37°C for 50 min, samples were subjected to 30 cycles of PCR (94°C for 45 s, 60°C for 45 s and 72°C for 30 s). The PCR products were separated on an ethidium bromide-stained 2% agarose gel.
For quantitative RTPCR cDNA concentration was adjusted at 100 ng/µl. Quantitative RTPCR was performed with a ready to use assay-on-demand kit for hTERT expression (Applied Biosystems, Hs00162669_m1) and an ABgene master mix kit (ABgene). Assays were performed in triplicates using an ABI 7700 QRTPCR system and carried out in 25 µl with 15 min preincubation at 95°C and 40 cycles of 45 s at 95°C and 60 s at 60°C. All data were normalized to GAPDH internal mRNA control (
CT analysis).
Methylation-specific PCR (MSP)
The methylation status of the hTERT-promoter was examined by MSP. Bisulfide modification of 1 µg DNA of all five cell types and MSP were performed as described previously (37). Primers for two CpG islands within the hTERT-promoter (region 1: 852 to 728, region 2: 635 to 438) were generated specific for unmethylated and methylated DNA (Table 1). PCRs were performed using 25 pmol/l primer, 25 µM dNTPs, 3 µl probe containing 0.1 µg bisulfide-treated DNA and the respective buffers. One unit of Taq-polymerase was added after performing a 5 min hot-start followed by 35 cycles of PCR [94°C for 30 s, 62°C (region 1) or 64°C (region 2) for 30 s, 72°C for 30 s]. Reactions were analyzed on ethidium bromide-stained 2% agarose gels.
Electrophoretic mobility shift assays (EMSAs)
Nuclear extracts from all five cell types were prepared as described previously (36,38,39).
-32P-labeled oligonucleotide DNA probes were constructed with 5 pmol of double-stranded oligonucleotides (Table II), synthesized by the phosphoramidite procedure, and purified by gel electrophoresis. Radiolabeling was done by a Klenow fill-in reaction (40). EMSAs with all oligonucleotides in all cell types were carried out by incubating 5 µg of nuclear extract with 5 fmol of the
-32P-labeled oligonucleotide DNA probe (20 000 c.p.m.) in a 20 µl binding reaction containing 50 mM TrisHCl, 10 mM MgCl2, 1 mM DTT, 1 mM EDTA, 16% glycerol and 1.0 µg of poly(dAdT) (Amersham Pharmacia Biotech). After incubation at 4°C for 45 min, the samples were loaded on a 6% polyacrylamide, 0.25x Trisborate gel and electrophoresed at 15 mA/gel (20 cm) for 2 h. The gels were dried and exposed to X-ray film (Kodak X-AR) at 80°C for 1216 h.
Competitor oligonucleotides and antibodies used in EMSAs and immune supershift reactions
For competition experiments, the nuclear extract was preincubated with 100-fold excess of unlabeled wild-type, mutant or consensus oligonucleotides prior to the addition of the
-32P-labeled probe. Immune supershift assays were performed using a monoclonal anti-p53 antibody (PAb 240, from Becton Dickinson), a polyclonal anti-c-myc antibody (N-262), a monoclonal anti-c-myc antibody (C-33), a polyclonal anti-E2F-1 antibody (C-20), a polyclonal anti-E2F-6 antibody (K-20) and a monoclonal anti-Sp1 antibody (1C6), respectively (all from Santa Cruz Biotechnology) (1:500). The antibodies were preincubated with the nuclear extract at 4°C for 12 h prior to the addition of the
-32P-labeled oligonucleotide DNA probe.
Western blotting
Lysates from all cell lines were prepared in a buffer (ELB) as described previously (33). Total protein samples (10 µg/sample) were separated on 610% SDSPAGE and proteins were transferred to a polyvinylidene difluoride membrane (Immobilon-P, Millipore). The membrane was blocked in 10% milk for 4 h at room temperature and incubated with a polyclonal anti-cyclin-D1 antibody (H 295, Santa Cruz Biotechnology), a monoclonal anti-pRb antibody (3H9, Becton Dickinson), a polyclonal ß-tubulin antibody (H 235, Santa Cruz Biotechnology) or the antibodies described above, respectively, overnight at 4°C, followed by incubation with either an anti-rabbit, an anti-mouse or an anti-goat horseradish peroxidase (HRP)-conjugated secondary antibody (Amersham Pharmacia Biotech) (1:3000) for 1 h at room temperature. The signal was visualized by an enhanced chemiluminescence system (ECL Plus, Amersham Pharmacia Biotech) and exposed to Kodak-X-Omat LS film.
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Results
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Generating genetically defined esophageal epithelial cells
To analyze the transcriptional regulation of the human telomerase subunit, hTERT, in the early steps of malignant transformation of esophageal squamous epithelial cells, we used five different cell types corresponding to normal, genetically altered mortal and immortal cells, by using genetic alterations frequently found in esophageal cancer as well as esophageal cancer cells. Normal primary esophageal epithelial cells, EPC (34) were retrovirally transduced using vectors containing either a dominant-negative p53, the human cyclin D1, or the human telomerase subunit hTERT. The generated cells used for further studies were designated as EPC-
p53, EPC-D1 and EPC-hTERT. Whereas primary EPC reached senescence after 4050 population doublings (PDs), EPC-
p53 ultimately senesced at 6080 PDs, as evidenced by growth arrest and enlargement of cells in culture. In contrast, the replicative life span of EPC-D1 and EPC-hTERT was significantly and reproducibly extended to over 400 PDs, consistent with immortalization (6). The esophageal cancer cell line TE-12 replicated faster, could be cultured up to 100% confluence and showed a higher PD rate per passage. Levels of endogenous and ectopically expressed cyclin D1, or dominant-negative p53 in parental EPC and infected cells as well as tumor cells were determined by western blot analysis (Figure 1).

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Fig. 1. Western blot analysis. Equal amounts of protein were separated on 6/10% SDSPAGE and western blot was performed. Primary antibodies [monoclonal anti-p53 antibody (PAb 240) and polyclonal anti-Cyclin-D1 antibody (H 296) (1:1000 each)] were incubated overnight at 4°C, followed by incubation with either an anti-rabbit or an anti-mouse HRP-conjugated secondary antibody.
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Telomere biology of the different esophageal epithelial cells
There was no significant telomerase activity to be detected in EPC and EPC-
p53 by TRAP-assay (Figure 2A). In contrast, the immortal EPC-D1 and EPC-hTERT cells as well as the cancer cell line TE-12 displayed robust telomerase activity. Telomere length determined by Southern blotting with a probe specific for mammalian telomeric repeats (TRF) revealed a progressive shortening of telomeres in EPC from 5 to 1.5 kb, which correlated with the respective PDs (Figure 2B). EPC-
p53 demonstrated similar kinetics but with a higher average telomere length, suggesting some telomerase activity in these cells, which might be below the detection level of the TRAP assay. In contrast, EPC-D1 and EPC-hTERT revealed a constant elongation of telomeres at a homogeneous length of 25 kb, correlating with their telomerase activity and immortalization. Interestingly, TE-12 cells revealed a very short but constant average telomere length between 1 and 2.5 kb, despite robust telomerase activity. RTPCR results showed hTERT expression in all five cell types, but to a lesser extent in EPC and EPC-
p53. Based upon quantitative RTPCR, we could only detect very low hTERT expression in EPC and EPC-
p53, whereas EPC-hTERT showed a 1.4-fold and EPC-D1 a 1.3-fold increase of hTERT-expression compared with TE-12 tumor cells, set as positive control in this assay (Figure 3).

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Fig. 2. Telomerase activity (TRAP) and telomere length (TRF). (A) Cellular extracts (100 ng) of all cell types were assayed for telomerase activity using the PCR-based TRAP-assay. Heat-treated (HT) samples served as negative control. IC is an internal PCR-control to demonstrate the absence of PCR inhibitors in the cellular extracts. (B) Telomere length for all cell lines was analyzed by hybridization of genomic DNA with a specific oligonucleotide probe. Different passages of cell lines are shown.
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Fig. 3. Quantitative real-time and RTPCR results. (A) Quantitative real time RTPCR was performed to detect relative levels of hTERT in EPC, EPC- p53, EPC-D1, EPC-hTERT and TE-12. (B) Relative expression levels of hTERT after QRTPCR. (C) RTPCR of hTERT in the respective cell lines. See online supplementary material for a colour version of this figure.
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Telomerase promoter activity is differentially regulated in esophageal epithelial cells
A 1242 bp hTERT-promoter construct designated as full length was subcloned in the promoterless pGL3 luciferase reporter plasmid and transiently transfected. Transfection efficiency between all cell types was similar, as SV40-promoter activity was uniform between the cell types. Additionally, the ratio of GFP positive cells remained stable within a given transfection experiment. The most prominent transcriptional activity was observed in TE-12, which showed an activity of 27.5% of the SV40-promoter. EPC cells reached 13% of this transcriptional activity, EPC-
p53 19%, EPC-D1 29% and EPC-hTERT 30%. Since the full-length hTERT-promoter proved to be sufficient to achieve gene expression, this region was subjected to functional analysis through deletion constructs. Deletion constructs (Figure 4F) containing 1011, 905, 700, 357, 290, 247, 187, 161, 101, 48 and 16 bp of the flanking DNA sequence 5' to the putative transcription start site were generated using a PCR-based strategy. All constructs were subcloned into the pGL3 basic luciferase reporter gene vector, and were used in a series of transient transfection experiments in EPC, EPC-
p53, EPC-D1, EPC-hTERT and TE-12 cells.

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Fig. 4. Transient transfections of the hTERT-promoter deletion constructs. hTERT-promoter luciferase reporter gene constructs (F) containing 1011, 905, 700, 357, 290, 247, 187, 161, 101, 48 and 16 bp of flanking DNA sequence 5' to the putative transcription start site were generated. All constructs were transiently transfected into EPC (A) EPC- p53 (B) EPC-hTERT (C) EPC-D1 (D) and TE-12 (E), and luciferase activity was measured. Luciferase activity is expressed as ratio of the 1242 bp full length hTERT-promoter (=100%) calculated from at least three independent transfection experiments (mean ± SE). Transfection efficiency was monitored using the pGL3-promoter plasmid and the GFP-expression vector pLEGFP-N1.
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The analysis revealed a stepwise decrease in promoter activity in all cells with the hTERT 1011 and 905 constructs, suggesting positive cis-regulatory elements upstream 905 bp. This decrease in promoter activity was less pronounced in TE-12 (Figure 4E). In TE-12 promoter activity did not change significantly until the 187 construct. With the 187 construct, a maximum of transcriptional activity was achieved. This region contains a well-described E-box of the hTERT core promoter. Downstream of this E-box telomerase promoter activity declined most significantly with the 161 and 48 constructs.
In contrast, in EPC (Figure 4A) a significant increase of hTERT-promoter activity was observed with the 700 construct, suggesting a negative cis-regulatory element further upstream. Promoter activity remained high up to the 161 construct. However, a substantial loss of promoter activity was observed with the 101 and 48 constructs. This indicates that the sequence between 161 and 48 bp contains cis-regulatory elements, which account for over 80% of hTERT basal promoter activity. This sequence contains multiple putative Sp1 binding sites.
After the initial positive cis-regulatory region, hTERT-promoter activity in EPC-
p53 cells showed a significant increase with the 700 construct, underscoring the presence of a negative cis-regulatory element (Figure 4B). With the 357, 290, 247, 187, and the 161 constructs, transcriptional activity remained mainly unchanged at a high level. Downstream, with the 101 construct, transcriptional activity declined deleting one putative Sp1 binding site.
In immortalized EPC-hTERT (Figure 4C), promoter activity increased significantly up to the 357 construct. With the 290 construct, activity declined correlating with a putative E2F binding site and increased again to more than twice the full length promoter activity with the 247 construct. Promoter activity then decreased, most dramatically between 161 and 101 bp of the hTERT core promoter.
In EPC-D1 (Figure 4D), activity significantly increased with the 700 construct indicating again the strong silencer region upstream of this promoter region. Further downstream, activity declined with the 290 construct indicating a strong positive cis-regulatory element within the 357 construct, which contains a putative E2F binding site. With the 247 construct, the hTERT-promoter activity achieved a second peak. The 290 construct upstream contains another putative E2F site. Downstream of 247 bp the promoter activity declined in a stepwise fashion corresponding to the deletion of an E-box and several Sp1 sites within the hTERT core promoter region.
The methylation status of the hTERT-promoter corresponds to an upstream silencer region
The hTERT-promoter is a GC-rich promoter and has three typical CpG islands within the first 1242 bp 5' of its ATG. Using the MethPrimer-Program (www.dahiyaurology.com/cgi-bin/methprimer.cgi), we selected two CpG islands upstream of the core-promoter, region 1 between 852 and 728 bp and region 2 between 635 bp and 438 bp, which potentially played an additional role in the regulation of hTERT (Table I). As shown in Figure 5, the hTERT promoter was methylated in region 1 in all the genetically altered cells as well as in tumor cells. Interestingly, only in EPC could we also detect an unmethylated hTERT promoter region. Region 2 remained unmethylated in all cell types (data not shown).

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Fig. 5. Methylation-specific PCR (MSP). MSP of an upstream region (852 to 728 bp) within the hTERT promoter was performed. The 5' and 3' primer are specific for unmethylated (u) or methylated (m) hTERT CpG island sequences.
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C-myc, Sp1 and E2F interact with the hTERT-promoter
Profound differences were observed in the transcriptional regulation of the hTERT core promoter (290 to 48). Within this core promoter region are multiple transcription factor binding sites. There are three putative Sp1 sites, two within the 101 construct, Sp1cWT(66) and Sp1bWT(99), and one within the 161 construct, Sp1aWT(121). Further upstream, within the 187 promoter construct is an E-box motif, E-boxWT(177) and within the 290 constructs are two putative E2F binding sites in both orientations, E2FWT(266), and E2FWT(281). An E-box located further downstream (+24 bp) seemed not to influence hTERT transcription in our particular cellular context. EMSAs were performed using nuclear extracts from all five different cell types with all potential transcription factor binding sites.
-32P-labeled double-stranded oligonucleotides representing, respectively, wild-type and mutant versions of each of the regions were used as probes (Table II). In addition, competition with double stranded wild-type, mutant or consensus oligonucleotides, as well as antibodies, determined the binding specificity of the respective putative transcription factors.
Transfection results in EPC cells suggested regulatory elements within the 161 and 101 constructs. Sp1 in EPC nuclear extract was found to bind specifically to all three potential Sp1 binding sites, as demonstrated by competition studies with excess consensus, mutant or wild-type oligonucleotides and antibody supershift experiments (Figure 6AC). Interestingly, in supershift assays with Sp1 and p53 antibodies at the Sp1cWT(66) oligonucleotide, the complex was abolished by an anti-p53 antibody, but not entirely by an anti-Sp1 antibody. In contrast, with the Sp1bWT(99) and Sp1aWT(121) oligonucleotides the complexes were abolished by anti-Sp1 antibodies. Unspecific IgG antibodies did not abolish any complex (data not shown). No specific binding could be observed using the E-box or the E2F-binding site containing oligonucleotides (Figure 6D and E).
In EPC-
p53 cells, only the upstream Sp1 element Sp1aWT(121) formed a specific Sp1-complex, confirmed by competition studies with excess consensus, mutant or wild-type oligonucleotides and supershift experiments (Figure 6C). This correlated with the transfection data where most of the promoter activity is located within the 161 construct that contains this particular Sp1aWT(121) element.
In EPC-hTERT cells, the 161 construct, and to a lesser extent, the 290 and 247 constructs appeared to regulate promoter activity. Within the first 161 bp the most apparent complex was found with the Sp1aWT(121) oligonucleotide, which could be eliminated by anti-Sp1 and anti-p53 antibodies (Figure 6C). In addition, transfection studies suggested a role for two potential E2F binding sites within the 290 constructs. EMSAs showed formation of specific complexes with the respective E2F oligonucleotides E2FWT(266) and E2FWT(281) (Figure 6D) but no specific complex could be observed with the E-box containing oligonucleotide (Figure 6E).
EPC-D1 cells revealed a more complex regulation involving the 161, 187, and 290 constructs, containing potential Sp1, c-myc and E2F binding sites. Analysis of EPC-D1 nuclear extract revealed a strong and highly specific Sp1-complex with the Sp1aWT(121) oligonucleotide (Figure 6C). Similar to EPC-hTERT cells, specific E2F complexes with E2FWT(266) and E2FWT(281) oligonucleotides could be demonstrated for EPC-D1 cell nuclear extracts. These complexes could be completely eliminated by an anti-E2F-1 antibody but only partially by an anti-E2F-6 antibody (Figure 6D). Additionally, we noted a specific complex with EPC-D1 nuclear extracts and the E-box containing oligonucleotide, E-boxWT(177) (Figure 6E). c-myc binding specificity was demonstrated by competition studies with excess mutant or wild-type oligonucleotides and competition in supershift experiments with the polyclonal anti-c-myc antibody (N-262). The monoclonal anti-c-myc antibody C-33 showed only weak competition.
Transcriptional regulation of hTERT-promoter in TE-12 appeared to be mainly regulated by this particular E-box motif. TE-12 nuclear extracts formed a specific complex with the E-boxWT(177) (Figure 6E). The polyclonal anti-c-myc antibody (N-262) was found to abolish the complex, suggesting c-myc is involved. Additional proteinDNA complexes were formed with oligonucleotides Sp1cWT(66) and Sp1bWT(99) (Figure 6A and B). This correlated with the transfection data.
Protein expression of involved transcription factors
All cell lines were assessed for pRb, E2F-1, c-myc and Sp1 expression. Hyperphosphorylated pRb was strongly and predominantly expressed in EPC-D1 and TE-12. Interestingly, there was also hyperphosphorylated pRB in EPC-
p53 cells. Hypophosphorylated pRb was predominantly expressed in EPC-hTERT cells. E2F-1 expression was found in all cell lines with an increase of expression in EPC-D1 and TE-12. c-myc and Sp1 expression was essentially constant with a slight increase of Sp1 in the primary EPC cells (Figure 7).

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Fig. 7. Western blot analysis of respective transcription factors. Equal amounts of protein were separated on 6/10% SDSPAGE and western blot was performed. Primary antibodies used were monoclonal anti-pRb (3H9), polyclonal anti-c-myc antibody (N-262), polyclonal anti-E2F-1 antibody (C-20), monoclonal anti-Sp1 antibody (1C6), and polyclonal ß-tubulin antibody (H 235) (1:500 each). Antibodies were incubated overnight at 4°C, followed by incubation with either an anti-rabbit, an anti-mouse or an anti-goat HRP-conjugated secondary antibody.
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Discussion
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Besides alterations in oncogenes and tumor suppressor genes, immortalization and transformation are associated with telomerase activation and accompanying telomere maintenance (4,41,42). A major mechanism to regulate telomerase activity is the transcriptional control of the catalytic subunit of the human telomerase gene, hTERT. It has been demonstrated that several transcription factors, including oncogene and tumor suppressor gene products are involved in the transcriptional regulation of human telomerase (19). Nevertheless, the majority of these studies have been performed in cancer cells, in which telomerase is already robustly activated. Since cancer cells bear a vast number of genetic alterations it is difficult to define which alterations play a causative role in hTERT regulation. This motivated us to determine the transcriptional regulation of human telomerase comparing normal cells with genetically altered, premalignant cells with cancer cells, using an experimental system in which we were able to analyze hTERT regulation under the influence of individual cancer related genetic alterations. To that end we studied mortal primary esophageal epithelial cells (EPC) and the generated derivates thereof harboring either a p53 inactivation (EPC-
p53), a cyclin D1 overexpression (EPC-D1) or an hTERT overexpression (EPC-hTERT) as well as esophageal squamous cancer cells (TE-12). We demonstrated that telomerase transcription and activity are differentially regulated in these cell types depending upon the specific genetic alteration observed in esophageal cancer. Whereas the telomerase promoter and subsequent telomerase expression in normal epithelial cells is mainly regulated by Sp1, the regulation in cells harboring cyclin D1 overexpression additionally involves c-myc and E2F. Interestingly, in cells with inactivated p53, Sp1 is also the main transcriptional regulator of hTERT promoter activity. In cells overexpressing hTERT, Sp1 and E2F play the most important roles in transcriptional regulation. Finally, in esophageal cancer cells, c-myc is mainly regulating the human telomerase promoter. This study demonstrates for the first time that human telomerase transcription and activity are differentially regulated in normal epithelial cells, versus genetically altered, mortal or immortal, premalignant cells versus the respective cancer cells. Furthermore, this differential regulation is mainly influenced by the specific genetic alteration involved. One other study by Casillas et al. (43) demonstrated changes in the transcriptional regulation of hTERT in the switch from normal WI-38 fibroblasts to their transformed counterparts. This study focused on the switch from mad-1/Max to c-myc/Max binding in SV40 T antigen, hTERT and ras transformed fibroblasts.
The zinc finger transcription factor Sp1 appeared to be the major regulator of hTERT transcription in normal EPC cells through multiple Sp1 sites within the hTERT core promoter (23,25,44). Nevertheless, it appears not to be sufficient to induce robust telomerase activity. Because of its ubiquitous expression in a wide range of normal as well as cancer cells Sp1 by itself seemed to be an unlikely candidate for differential hTERT transcriptional regulation on the way from normal to cancer cells. Interestingly, we observed essentially similar levels of Sp1 in all our cell lines, regardless of hTERT promoter activity. Therefore, the protein level of Sp1 does not seem to play the major role in Sp1 mediated transcriptional regulation. It has been described, that hTERT expressing cells as well as telomerase negative cells can have similar levels of Sp1 binding to the hTERT-promoter (45). Sp1 has also been shown to recruit basic transcription factors and thereby play a significant role in transcriptional initiation of TATA-less promoters as the hTERT-promoter (46). Those mechanisms could be operative in EPC cells as well establishing a critical role of Sp1 in hTERT basal transcriptional activity in a respective cellular context. In our study Sp1 is also involved in the transcriptional regulation of EPC-
p53, immortal EPC-D1 and EPC-hTERT as well as the esophageal cancer cells TE-12, although mediated through different Sp1 responsive elements. In these different settings including a cancerous state Sp1's interaction with other proteins, transcription factors like p53 or myc or corepressors like histone deacetylases (HDAC) may positively or negatively regulate hTERT transcription (27,47,48). We have demonstrated such a possible interaction with p53 in our EMSA analysis. Therefore, inactivation of interacting proteins e.g. through deletion of their binding site may allow Sp1 to function as a transcriptional activator of the telomerase promoter, as we could demonstrate for EPC and EPC-
p53. As a tumor suppressor p53 is frequently mutated in human esophageal cancer, and it has been discussed that one mode of p53's tumor suppressor function might be repression of hTERT through interaction with Sp1 (47,49). Nevertheless, it remains unlikely that p53 inactivation plays the major causative role in telomerase activation during carcinogenesis, since telomerase activity remains undetectable in EPC-
p53 cells despite p53 inactivation. Furthermore, these cells remain mortal. Interestingly, EPC-
p53 cells show hTERT expression and promoter activity but no telomerase activity. Since telomere length in EPC-
p53 is longer than in primary EPC, there might be some residual telomerase activity above that of normal cells (11) not picked up by the TRAP-assay.
The fact that in a tumor cell the hTERT promoter is predominantly activated by c-myc confirms many previous studies in cancer cells in which c-myc has been shown to bind E-boxes within the hTERT core promoter and to activate hTERT transcription (30,50). In general, the transcriptional regulation through these E-boxes seems to be mediated by the complex formation of either c-myc/Max versus Mad/Max determining whether the core promoter is activated or repressed (29). In our experiments, we did not observe any appreciable differences in c-myc protein level regardless of its involvement in hTERT promoter regulation in a respective cell line. Therefore, hTERT transcriptional regulation by c-myc seems not to depend on c-myc levels in a respective cell. Interestingly, in some experimental systems the control of hTERT transcription depends on the E-box element but also does not correlate with the amount of endogenous c-myc protein (51,52), confirming our results. In our experimental setting only the upstream E-box located at 177 bp played a role in activating the hTERT promoter. There is another E-box located further downstream at position +24 bp. This E-box is described to induce hTERT expression in different tumor cells, but is not functional in our cellular context.
The E2F family of transcription factors generally transactivates genes involved in cell cycle progression. We observed multiple important E2F responsive elements within the hTERT promoter and were able to demonstrate that some of these E2F binding sites are positive and others negative cis-regulatory elements of the hTERT-promoter. For the negative cis-regulatory elements E2FWT(266) and E2FWT(281), which play an important role in EPC-D1 and EPC-hTERT, we demonstrated E2F-1 binding, confirming its role as a repressor in a premalignant cell. Some data already indicated that E2F-1 potentially repress the hTERT-promoter in tumor cells while inducing the hTERT-promoter in normal cells (53,54). Interestingly, E2F-1 seemed not to play a role in hTERT transcriptional regulation in the esophageal cancer cell line TE-12, although we could demonstrate a substantial pRB hyperphosphorylation in these cells.
Besides the respective genetic alteration, the state of mortality of the respective cell type might influence the transcriptional mechanisms regulating hTERT transcription as well. The mortal cell types, primary EPC as well as EPC-
p53 were predominantly regulated by Sp1, as discussed, whereas the immortal cell types EPC-D1 and EPC-hTERT demonstrated more complex regulatory mechanisms. In this regard it is not clear why cyclin D1 overexpressing cells demonstrate telomerase reactivation and an immortal phenotype. Most likely EPC-D1 acquired secondary genetic alterations leading to telomerase activity and immortalization.
Another mechanism regulating hTERT transcription might be hypermethylation of its promoter. The presence of CpG islands upstream of the hTERT core promoter prompted us to examine a possible role of promoter methylation in the hTERT regulation of normal cells, genetically altered cells and cancer cells. Interestingly, one region was methylated in the immortalized cell types EPC-D1 and EPC-hTERT, the cancer cells TE-12 as well as the mortal cell type EPC-
p53. Notably, this region showed a partly unmethylated status in primary EPC cells. The fact that normal cells without detectable telomerase activity show both methylated and unmethylated sites indicates that other mechanisms independent of DNA methylation must repress telomerase activity in normal cells and confirms previous studies (55). Furthermore, these results correlate well with one recent study, which found this region to be only partially methylated in normal tissues and fully methylated in tumorous tissues (56). The methylated region in the genetically altered cells at least correlated with a negative cis-acting element between 700 and 905 bp of the hTERT promoter and could contribute to its regulation. Obviously we appreciate that one cannot directly link promoter regulation measured in transient transfection analyses and epigenetic regulation via promoter methylation. Nevertheless, our findings would suggest that promoter methylation is involved in hTERT regulation of cells altered by cancer related genes potentially through the silencer region 700 to 905 bp. Other epigenetic mechanisms might also be involved in hTERT transcriptional regulation during carcinogenesis. Recent data suggested that the endogenous chromatin environment might play a critical role in the regulation of hTERT expression during cellular immortalization (48).
Our model of genetically defined cells representing important genetic alterations as they are seen in esophageal carcinogenesis allowed a detailed analysis of hTERT regulation influenced by these different alterations. We demonstrated for the first time that telomerase transcriptional regulation and subsequent activation underlies complex regulatory mechanisms, and is differentially regulated when comparing normal cells, genetically altered, premalignant esophageal cells and esophageal cancer cells. The transcriptional regulation is mediated by Sp1, E2F and c-mycall binding to their respective sites within the core promoter of hTERTbut the methylation status of the promoter might play a role during this complex regulatory process as well. A detailed analysis of each individual mechanism in this process is now necessary. Nevertheless, our novel data provide new insight into the regulatory mechanisms of telomerase transcription and activity in early steps of esophageal carcinogenesis.
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Supplementary material
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Supplementary material can be found at: http://carcin.oxfordjournals.org/
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Acknowledgments
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We are grateful to S.A.Reeves and R.A.Weinberg for the cyclin D1 and hTERT constructs, respectively, to A. Sosnowski and C. Arnold for their technical assistance with the methylation specific PCR and to the Rustgi and Opitz labs for discussion. This work was supported by grants from the Deutsche Krebshilfe (10-1656-Op 1 and 10-2209-Op 2).
Conflict of Interest Statement: None declared.
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Received December 22, 2004;
revised May 17, 2005;
accepted June 7, 2005.