1 Institut de Pathologie, Centre Hospitalier Universitaire Vaudois, CH-1011 Lausanne, Switzerland and
2 Institut Bergonié, 33076 Bordeaux, France
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Abstract |
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Abbreviations: ALT, alternative lengthening of telomeres; MS-SSCA, methylation-specific single stand conformation analysis; STS, soft tissue sarcomas.
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Introduction |
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The hTERC gene was identified a few years ago (8). Since most RTPCR-based experiments showed that this RNA was widely expressed in both tumoral and non-tumoral tissues (9,10), it was concluded that hTERC was not essential to telomerase reactivation. However, the examination of a series of tumors using in situ hybridization revealed an up-regulation of hTERC expression in tumor tissues (11,12). Weak expression was also occasionally detected in some normal tissues, namely gastric, esophageal and prostate epithelial basal layers, and activated lymphocytes (1214). In a previous study, we demonstrated that hTERC expression was closely linked to telomerase activity in colorectal carcinogenesis (15) suggesting that this gene could play a role during the process of telomerase re-activation. Based on these observations, we thought it would be interesting to see if hTERC regulation could be a tumor-specific phenomenon. Characterization of the human TR gene revealed that several sites might be involved in its regulation (8). In a recent report, Zhao et al. (16) showed that the hTERC gene may be activated by the transcription complex NF-Y, also by transcription factors such as Sp1 and pRB (Retinoblastoma protein), and may be repressed by Sp3. Furthermore, the presence of a large CpG island within the hTERC gene suggests that methylation could be implicated in hTERC regulation as well. Recently, a strong correlation between hTERC promoter methylation and lack of hTERC expression was observed exclusively in telomerase-negative cell lines (17). Therefore, it is possible that hTERC methylation results in hTERC silencing in at least a subset of telomerase-negative tumors.
Immortalized telomerase-negative tumors are malignant cells, which use a telomerase-independent mechanism (18). A subset of this tumor category, using the ALT mechanism (alternative lengthening of telomeres) (19) has been shown to exhibit ultra-long, heterogeneously sized telomeres and characteristic multiprotein structures (20). Recent studies suggested that the length of telomeres in ALT cells might be obtained by homologous recombination and copies switching between telomeric tracts (21,22). Soft tissue sarcomas (STS) constitute a large and heterogeneous group of malignant mesenchymal tumors. About half of them do not express telomerase (23,24). These telomerase-negative STS might constitute an appropriate material for studying the regulation of hTERC expression.
In the present study, we examined hTERC expression and hTERC methylation in a series of human normal tissues, non-soft tissue tumors, and tumor cell lines, as well as in a series of telomerase-negative STS. We observed strong variations of hTERC expression according to the cell type studied. In addition, the analysis of methylation patterns showed that the hTERC gene is unlikely to be regulated by methylation-based mechanisms in telomerase-negative normal tissues and in tumor tissues. The few partial methylation patterns observed in telomerase-positive cell lines could represent a side effect of cell culture.
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Materials and methods |
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Tumor cell lines
Ten human tumor cell lines (breast, MCF-7; cervix, A431, HeLa; colon, Co115, SW480; lung, H520, SW2; prostate, PC-3; osteosarcoma, Saos-2, U-2 os) were also studied. With few exceptions (SW480, SW2 and Co115, from the Swiss Institute for Cancer Research, ISREC, Lausanne, Switzerland), these cells were obtained from the American Type Culture Collection. Cells were routinely cultured in Dulbecco's modified medium with glutamax-1 supplemented with 10% fetal bovine serum (5% for Saos-2 and U-2 os), or Leibovitz medium (L15) with 5% FBS and 0.2% NaHCO3 for SW480 (all products from Gibco BRL, Paisley, UK). All cells lines were tested and found to be negative for Mycoplasma contamination.
DNA and RNA extraction and TRAP assay
DNA, RNA and proteins were extracted from consecutive tissue sections. To establish the methylation status of hTERC promoter and exon, genomic DNA was isolated using the DNeasy tissue kit (Qiagen, Germany) according to the manufacturer's protocol. Total RNA was extracted from frozen tissue sections or cells using Trizol (Life Technologies, Rockville, MD). RTPCR on hTERT RNA was performed as described previously (23). TRAP assay was performed according to the modified protocol described by Yan et al. (23).
Analysis of hTERC expression by RTPCR and quantitative dot blot
Total RNA (2.5 µg) was first digested by DNase I (Roche Diagnostics, Mannheim, Germany) according to the manufacturer's protocol. RNA was purified by phenolchloroform extraction followed by ethanol precipitation. cDNA was obtained using pd(N)6 random primer (Amersham, Freiburg, Germany) and Expand Reverse Transcriptase (Roche Diagnostics) as per manufacturer's protocol. PCR on hTERC cDNA was performed by using the primer set 5'-CGCCGTGCTTTTGCTCC-3' and 5'-ACTCGCTCCGTTCCTCTTCC-3', in a final 5% DMSO, and the following PCR conditions: 23 cycles of 94°C for 30 s, 62°C for 45 s and 72°C for 45 s, followed by 10 min at 72°C on a Primus (MWG-Biotech) apparatus. cDNA quality was checked by PCR amplification of p53 and GAPDH cDNA (23). Calibration scales were realized by mixing total RNA from an hTERC positive cell line (HeLa) with total RNA from a negative sample (U-2 os) in 100 ng final. Percentages used were: 100, 50, 25, 10, 2.5, 1, 0.25 and 0%. Then, RTPCR was realized in the conditions described above. All tissue samples and calibration scales were amplified together in the same PCR reactions. A DIG-labeled probe was produced by re-amplification of an hTERC positive RTPCR, in a PCR including dUTP-DIG.
For quantitative dot blot, RTPCR products were denatured 10 min at 100°C and put immediately on ice. Two microliters of each product were loaded on Eletran® N+ nylon membrane (BDH Laboratory Supplies, Poole, UK) and fixed under UV. The membrane was pre-hybridized in a 5x SSC/2x blocking/0.02% SDS/0.1% N-lauroyl/50% formamide for 1 h at 42°C. Hybridization with the DIG-labeled probe (100 ng in 3 ml) was done for 2 h at 42°C. The membrane was then washed twice in 2x SSC/0.1% SDS for 5 min at room temperature, and twice in 0.2x SSC/0.1% SDS for 15 min at 68°C. After 2 min incubation in maleate buffer pH 7.5/Tween® 20, and 30 min in maleate buffer pH 7.5/1x blocking, the antibody anti-DIG (Roche Diagnostics) was added. The chemiluminescence reaction was performed after three washes with maleate buffer pH 7.5/Tween® 20, and one wash in 100 mM Tris pH 9.5/100 mM NaCl. Detection was realized with CDP-Star+ ready-to-use (Roche Diagnostics) according to the manufacturer's protocol. Signals were analyzed from X-omat film (Eastman Kodak Company, Rochester, NY) after different times of exposure, by comparing intensities with the internal calibration scale.
hTERC methylation analysis by MS-SSCA and sequencing after bisulfite modification
In order to differentiate methylated from unmethylated cytosine, genomic DNA was modified by sodium bisulfite using a protocol adapted from Raizis et al. (26) and Bian et al. (27). Two microliters of DNA in 36 µl of water were cleaved by 4 µl of 1 N HCl for 2 min exactly at room temperature. Then, 4.5 µl of 3 M NaOH was added and DNA denaturation was performed for 20 min at 37°C. Sodium bisulfite (500 µl) and hydroxyquinone (28 µl) were then added to a final concentration of 40.5% and 10 mM, respectively. The reaction was performed overnight at 55°C. After addition of 80 µl of water and 365 µ1 of pure ethanol, DNA was purified using the DNeasy tissue kit columns (Qiagen). Following washing with the kit wash buffer, desulfonation was performed on the column by addition of 500 µl of 0.15 M NaOH I 90% EtOH. Incubation was performed for 10 min at room temperature and in the dark. After washing, the modified DNA was eluted from the column with 50 µl of 10 mM of TrisHCl pH 8.0.
Two sets of primers were used for PCR on hTERC, one for the promoter region: 5'-GGAAATGGAATTTTAATTTTT-3' and 5'-AACCAACAACTAACATTTTTT-3', and one for the exon region: 5'-TAAATAAAAAATGTTAGTTGT-3' and 5'-ACCTAAAAAACCTAAACC-3'. PCR conditions used to amplify hTERC promoter were 40 cycles of 94°C for 45 s, 51°C for 45 s and 72°C for 75 s, followed by 15 min at 72°C. The same PCR cycling conditions in a final concentration of 5% DMSO and an annealing temperature of 48°C were used for hTERC exon. Sequencing of all PCR products was done on an ABI prism 310 sequencer (Perkin-Elmer, Branchburg, NJ). Each PCR product was analyzed by methylation-specific single stand conformation analysis (MS-SSCA) as described previously (27).
Control plasmid was generated by subcloning 672 bp, bases 212 to +459 bp of the hTERC gene (GenBank accession no. U86046), in the pGEM-T vector (Promega, Madison, WI). The plasmid was divided in two parts: one was left unmethylated and the other was fully methylated at all CpG sites using SssI methylase (New England Biolabs, Hertfordshire, UK) according to the manufacturer's protocol. Unmethylated and methylated plasmids were mixed at different ratios. The bisulfite modification was performed on fully methylated and unmethylated plasmids, as well as on different mixes.
5-aza-dC treatment
Cells were immediately treated after seeding in standard conditions, with 3 µM of 5-aza-dC every 48 h for 1 week. The cells were then collected for DNA and RNA extraction.
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Results |
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hTERC expression and methylation in telomerase-negative soft tissue sarcomas
Twenty-two telomerase-negative STS were analyzed in this study. hTERC expression showed wide variations (range from 0 to 186%) (Table II ). Interestingly, half of the tumors (11/22) expressed hTERC at a level similar to that of normal tissues (between 0 and 6%) (Figure 2
), including four of the five liposarcomas and three of the five leiomyosarcomas examined. In 41% (9/22) of the telomerase-negative STS, the hTERC expression level was similar to that observed in the telomerase-positive tumors (1070%) (Table II
). Only two samples showed an expression as strong as that of the cell lines (
70%). Methylation status was analyzed by MS-SSCA and by sequencing after bisulfite modification in all these telomerase-negative STS (Figure 1
, lanes 710). No methylation could be detected in any of them.
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A previous study revealed that telomere length was heterogeneous and did not correlate with telomerase activity (25). In the present report, we tried to correlate hTERC expression with telomere length in the 22 telomerase-negative STS. TRF fragments were assessed as short (S), medium (M) or long (L) when the medium size was situated under 9.6 kb (20% shorter than mean normal skeletal muscle TRF length), between 9.6 and 14.4 kb, and over 14.4 kb, respectively (25). In our series, samples were evenly distributed: seven small, eight medium and seven long. Means of hTERC expressions were 20, 26 and 21% (relative to the 100% in HeLa cells) for short, medium and long telomeres, respectively.
Finally, we tried to correlate hTERC and hTERT expressions. As shown in Table III, most telomerase-negative tumors expressed hTERC but not hTERT (16/22, 73%). Surprisingly, three cases expressed both genes, without any detectable telomerase activity. Finally, only three cases (one MPNST and two pleomorphic liposarcomas) were found that presented no hTERC nor hTERT expression (Table II
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Discussion |
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In order to answer these questions, hTERC expression was quantified by dot blot after RTPCR in a series of 60 samples (Tables I and II). As hTERC gene does not contain any intron, DNase I digestion was an absolute requirement to avoid a possible contamination of the results with genomic DNA. This quantitative PCR and dot blot showed that most normal human tissues did not express hTERC RNA. Although a few normal cases revealed weak transcription, the mean of hTERC expression was at least 30 times less in normal tissues than in telomerase-positive tumor tissues and 250 times less than in telomerase-positive tumor cell lines. This strongly suggests that hTERC gene is strongly up-regulated during tumorigenesis. A similar ratio in hTERT re-expression (about 24 times) has been observed between normal renal tissue and sporadic renal cell carcinoma (28). Thus, it appears that both hTERT and hTERC seem to be induced or strongly up-regulated during carcinogenesis. Therefore, as hTERT, hTERC expression can be considered as a marker of cell transformation. Few studies have tried to quantify and compare hTERC and hTERT RNAs in the same tumor tissue sample. In three types of human cancer (gliomas, hepatocarcinomas and breast carcinomas), a linear relationship between both RNA types was observed when hTERT expression reached a certain level of expression. In in situ carcinoma of the uterine cervix, a linear relationship between hTERC RNA and hTERT mRNA could be defined whereas such a correlation was not observed in precancerous dysplasia of the cervix (29). Therefore, transcriptional levels of both genes might be cross-regulated when the cancer becomes established.
hTERC expression was also investigated in telomerase-negative tumor cell lines and in tumor tissues. hTERC RNA was not detected in the tumor cell lines, Saos-2 and U-2 os. In contrast, marked variations (0150%) of hTERC expression were observed in the group composed of 22 telomerase-negative STS (Table II). In comparison with the hTERC levels observed in telomerase-negative normal tissues and in telomerase-positive tumor tissues, STS samples could be divided in to two groups: the first group, comprising half the samples, showed a level of hTERC expression similar to normal tissue, whereas the other half showed levels similar to the telomerase-positive tumors (Figure 2
). Therefore, hTERC expression could not be used as a tumor marker in soft tissue sarcoma.
The presence of a CpG-rich region in the promoter and in the exon of hTERC led us to hypothesize that it could be one of the putative hTERC regulatory elements. In a recent study, Hoare et al. (17) examined the role of methylation in hTERC transcription. Their results indicated that methylation might be implicated in telomerase-negative cell lines, but not in telomerase-negative normal tissues nor in telomerase-positive tumor tissues. In the present study, using direct sequencing and MS-SSCA after bisulfite modification of genomic DNA, identical results were obtained in normal tissue and tumor samples of various histology and location. MS-SSCA allows a clonal analysis of DNA population mixture where any clone >510% can be easily detected (27). As all tumor samples contained at least 70% of tumor cells, it is probable that hTERC methylation is not implicated in hTERC transcription fluctuations. Interestingly, three of the eight telomerase-positive cell lines (HeLa, MCF-7 and A431) showed a hypermethylated pattern by sequencing. Similar results were obtained for HeLa and MCF-7 (17). In the present study, the use of MS-SSCA allowed us to demonstrate that one allele was fully methylated whereas the other one was fully unmethylated, indicating that one allele is sufficient to induce hTERC gene transcription (Figure 1).
We next examined hTERC methylation patterns in 22 telomerase-negative STS. In contrast to the two telomerase-negative cell lines which were found to be hypermethylated, hTERC was not methylated in this series of 22 tumors, suggesting that methylation is not important in the hTERC regulation mechanism within this interesting group of telomerase-negative tumors.
How can we explain the occurrence of hTERC methylation in some of the analyzed tumor cell lines? Aberrant methylation has already been reported in cultured normal fibroblasts (30). In this particular experiment, growth constraints altered CpG island methylation, leading to alterations in epigenetic stability. Furthermore, we observed that treatment with 5-aza-dC of the telomerase-negative cell line U-2 os did not lead to the re-expression of the hTERC gene. Thus, the hTERC methylation detected in the two telomerase-negative cell lines as well as in the three telomerase-positive cell lines might merely correspond to a non-specific side effect of cell culture.
Recently, in situ hybridization performed on STS suggested that telomerase RNA expression may be up-regulated in tumor cells and may precede morphological transduction (31). In the present study, a huge range of hTERC expressions were obtained (from 0 to 186%), indicating that hTERC RNA is not an appropriate tumoral marker for STS, whereas its use might be relevant for epithelial tumors. Further studies will be necessary to investigate this hypothesis. In telomerase-negative STS, we tried to correlate hTERC expression with several other parameters, such as the cell proliferation rate as assessed by Mib-1 staining, telomere length and hTERT expression in the group of telomerase-negative STS. Our results indicate that hTERC expression does not correlate with Mib-1 staining or telomere length. Therefore, other parameters, which could interfere with hTERC transcription have to be identified and studied. In a recent report, Zhao et al. (16) showed that hTERC might be activated by the transcription complex NF-Y, by the transcription factors Sp1 and pRB (retinoblastoma) and could be repressed by Sp3. Such transcription factors might play a role in the variations observed in hTERC transcription. On the other hand, we found that most telomerase-negative STS did not express hTERT, suggesting that the lack of telomerase activity probably came from the lack of hTERT transcription. Along the same lines, we failed to find a tumor in our series, which expressed hTERT but not hTERC. Interestingly, three samples lacked expression of both genes, whereas three others did not show any telomerase activity in spite of expressing both genes. This latter finding lends weight to the notion that not only telomerase genes expression but also post-transcriptional modifications are required to obtain telomerase activity (32).
In summary, the present study shows that the levels of hTERC expression may vary according to the cell type examined (normal tissue versus telomerase-positive tumor versus telomerase-positive tumor cell lines) and are up-regulated during tumorigenesis. With the exception of telomerase-negative cell lines, variations in hTERC RNA expression are not related to a change in the methylation status of the hTERC gene. In addition, variations in hTERC expression in telomerase-negative STS were not linked to the methylation status, proliferation rate, telomere length, or hTERT expression.
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Notes |
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* These authors contributed equally to this work.
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Acknowledgments |
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References |
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