Japanese Journal of Clinical Oncology 32:3-8 (2002)
© 2002 Foundation for Promotion of Cancer Research
Methylation Status and Expression of Human Telomerase Reverse Transcriptase mRNA in Relation to Hypermethylation of the p16 gene in Colorectal Cancers as Analyzed by Bisulfite PCR-SSCP
1Division of Clinical Laboratory, 2Division of Surgery and 3Director, National Cancer Center Hospital, Tokyo, Japan
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ABSTRACT |
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Background: The expression level of human telomerase reverse transcriptase (hTERT) is correlated with telomerase activity and is expressed at high levels in malignant tumors. It is of interest whether expression of hTERT is regulated by methylation of the CpG island in the promoter of the hTERT gene. We examined hTERT expression and methylation status of the hTERT and other genes including p16.
Methods: We analyzed methylation status by bisulfite treatment and polymerase chain reaction with single-strand conformation polymorphism analysis (PCR-SSCP) and expression of the hTERT by RT-PCR, in 13 cancer cell lines, eight white blood cell samples and 24 colorectal cancer tissues.
Results: In the cancer cell lines, hTERT was expressed and the CpG island of the hTERT promoter was methylated. Most colorectal cancer tissues showed similar results. The promoter of hTERT was methylated in six cases, partially methylated in 17 cases and unmethylated in one case. All cases with methylation of hMLH1 or p16 also showed methylation of hTERT; however, some of the cases lacking p16 methylation also had hTERT methylation.
Conclusion: Increased expression of hTERT is related to hypermethylation of hTERT in colorectal cancerous tissues as well as some cancer cell lines and disconcordant with hypermethylation of p16.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
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Regulation of expression of the telomerase catalytic subunit, human telomerase reverse transcriptase (hTERT), is considered the major determinant of enzyme activity (1,2). hTERT is expressed at high levels in malignant tumors and cancer cell lines but not in normal tissues or telomerase-negative cell lines. A close correlation has been found between hTERT expression and telomerase activity in a variety of tumors (3–6). The 5'-regulatory region of the hTERT gene was recently cloned and characterized (7–10). The hTERT gene promoter lacks a TATA or CCAAT box, but a number of potential transcription factor binding sites, including potential binding sites for SP1, MAZ (Myc-associated zinc finger protein), a bHLHZ class of transcription factors (E-boxes), c-Ets-2 and AP-2 (7–10) are present. Luciferase assays assessing promoter activity revealed that a 59-bp region (–208 to –150) is required for maximal promoter activity (7). The 5' end of the hTERT cDNA contains GC-rich sequences, indicating the presence of a CpG island. hTERT expression may be regulated by methylation.
Promoter methylation plays an important role in the regulation of gene transcription, X chromosome inactivation, genomic imprinting and carcinogenesis (11–13). CpG islands are found within the promoter regions of ~60% of human genes (14) and these CpG islands normally are not methylated regardless of the expression status of the genes. Aberrant DNA methylation is an important alternative mechanism for mutations in coding regions that lead to inactivation of tumor suppressor genes and mismatch repair genes during neoplasia (12,13,15). It is of interest whether expression of hTERT is also regulated by methylation of the CpG island in the promoter of the hTERT gene. We hypothesized that the hTERT CpG island would be unmethylated to permit expression of hTERT in most telomerase-positive cells. Data from two previous reports, however, showed that the hTERT promoter region was mostly methylated and that hTERT was expressed in some cancer cell lines (16,17). Also, only a few clinical cancer specimens were examined (17). Here we tried to investigate the methylation status of a portion of the promoter of the hTERT gene in some colorectal cancerous tissues as well as some cancer cell lines, using bisulfite treatment and polymerase chain reaction with single-strand conformation polymorphism analysis (PCR-SSCP) (BiPS) (18). We also examined the association between expression of hTERT mRNA and methylation status of genes including p16, hMLH1 and HIC1 in colorectal cancer tissues.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
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Subjects
Thirteen tumor cell lines were provided as subjects. MKN1, MKN7, MKN28, MKN45, MKN74, KATO III (Japanese Cell Resource Bank) and NEDATE (National Cancer Center Hospital) cell lines were derived from stomach cancers. SW1116 (American Type Culture Collection), C-1 and Colo320HSR cell lines (National Cancer Center Research Institute) were derived from colorectal cancers. PSN1, Lu65 and A431 cell lines (National Cancer Center Research Institute) derived from pancreas, lung and vulva cancers, respectively, were also used. Colorectal cancer tissues, normal accompanying mucosa or tissue and metastatic lesions of liver and normal liver tissue resected at surgery from 24 patients were obtained from the National Cancer Center Hospital, Japan. Experimental use of specimens in addition to pathological examination was consented to by each patient. Peripheral white blood cells (PWB) were obtained from eight normal volunteers.
DNA Extraction, RNA Preparation and cDNA Synthesis
DNA was extracted from the tissue specimens according to a method described previously (19). Total RNA was prepared from the various cancer cell lines, and the PWB, the colorectal cancer tissues and corresponding normal mucosa by a standard guanidium thiocyanate–phenol–chloroform–isoamyl alcohol extraction method (20). Approximately 0.5–1.0 µg of total RNA was reverse transcribed with random hexamers as primers and SuperScript RNase H reverse transcriptase (GIBCO BRL, Gaithersburg, MD).
BiPS Analysis
The BiPS procedure was performed as described previously (18). Briefly, bisulfite treatment was done with reagents provided in the Oncor CpGenome DNA Modification Kit (Intergen, Purchase, NY). Control methylated DNA was prepared with SssI methylase (New England Biolabs, Tokyo, Japan) from genomic DNA of PWB. PCR primers were designed to be complementary to the chemically modified DNA with no CpG sites in the corresponding region of the genomic DNA. The primer sequences for amplification of hTERT were 5'-GGGTTTTTAGTGGATT-3' (sense) and 5'-AAACTAAAAAATAAAAAAACAAAAC-3' (antisense). The primer sequences corresponded to nucleotide positions 1432–1447 and 1536–1512, respectively, of the hTERT gene (GenBank AF098956). The region selected for amplification has been related to the promoter activity (7–10). The expected size of the RT-PCR product was 105 basepairs containing nine CpG sites. PCR was performed with AmpliTaq Gold (PE Applied Biosystems, Branchburg, NJ) and the hot start procedure. SSCP analysis was performed using 15% non-denaturing polyacrylamide gel and silver staining detection (Daiichi Pure Chemicals, Tokyo, Japan).
Sequencing Reaction of the Methylated DNAs
Extra bands (possibly methylated) detected by BiPS analysis were excised from the gels and reamplified. The PCR products were treated with shrimp alkaline phosphatase and exonuclease III (Amersham Pharmacia Biotech, Amersham, Bucks, UK) to remove excess PCR primers and nucleotides and then sequenced directly by the dideoxy sequencing procedure with a BigDye Terminator Cycle Sequencing FS Ready Reaction Kit and ABI PRISM 310 Genetic Analyzer (PE Applied Biosystems).
Assessment of hTERT mRNA Levels by RT-PCR
Expression of hTERT was analyzed by RT-PCR with the primers described by Nakamura et al. (2). ß-Actin mRNA was amplified as described (21) for internal control. PCR products were separated by electrophoresis on 8% polyacrylamide gels and visualized by ethidium bromide staining and UV illumination.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
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Cancer Cell Lines and Peripheral White Blood Cells
According to RT-PCR analysis, ß-actin and hTERT were expressed in all 13 cell lines examined (Fig. 1). In PWB, ß-actin was expressed in all eight samples, whereas no or only trace amounts of the expected length of hTERT were observed. With BiPS analysis, 11 cell lines showed three bands that migrated to the same positions as those of fully methylated control DNAs (Fig. 2). One cell line, MKN28, yielded four bands. Although one of the bands migrated to a similar position as the unmethylated control DNA, it was identified unexpectedly as methylated. MKN74 was not informative owing to unsuccessful PCR amplification. All PWB samples showed bands that migrated to the same position as the unmethylated control DNA bands (Fig. 2).
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Colorectal Cancer Tissues
In normal mucosa, hTERT expression was negative in 18 cases, weakly positive in three cases and positive in three cases (data not shown). In cancerous tissues, hTERT expression was negative in three cases and positive in 21 cases.
We classified the BiPS patterns of cancerous tissues. The hTERT gene promoter was methylated in six cases, partially methylated in 17 cases and mostly unmethylated in one case (Fig. 3). Sequence analysis of the amplified products revealed complex methylation patterns. Methylation patterns for hTERT consisted of mixtures of unmethylated and methylated DNA at each CpG site (Fig. 4). In Case No. 147, we were able to analyze metastatic foci as well as the primary tumor and found that normal mucosa and metastatic liver tissue had mixed patterns of methylated and unmethylated bands; cancerous tissue showed only methylated bands and normal liver tissue showed only unmethylated bands. This may be due to differences in the methylation status between organs or the cell population analyzed.
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Relation Between Methylation Status or Expression of Htert Gene and Other Genetic Properties
Nearly all colorectal cancerous tissues analyzed showed aberrant methylation of hTERT gene (Table 1). Of the 24 cases, seven, five and 20 cases showed methylation of hMLH1, p16 and HIC1, respectively (15). All cases with methylation of hMLH1 or p16 also showed hTERT methylation. All cases with microsatellite instability also showed hTERT methylation. The one case without hTERT methylation was one of four cases lacking methylation of HIC1 gene.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
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We had hypothesized that the hTERT CpG island would be unmethylated to permit expression of hTERT in telomerase-positive cells. Our present data and data from two previous reports (16,17), however, showed that the hTERT promoter is methylated and that hTERT is expressed in some cancer cell lines and cancer specimens. Methylation of the CpG island in the hTERT gene promoter appears not to be responsible for direct repression of hTERT expression.
5-Aza-2'-deoxycytidine (5-aza-CR), a demethylating agent, caused down-regulation of hTERT expression in several cancer cell lines (16,22). This alteration in expression levels may be influenced by the cell cycle regulator protein p16 and the p16 gene is silenced by hypermethylation in some cancer tissues and cancer cell lines. Kitagawa et al. (22) indicated that up-regulation of p16 and subsequent down-regulation of c-myc are major pathways for hTERT repression by 5-aza-CR. It has been reported that myc proteins activate hTERT transcription directly through the E-box located within the core promoter (7,9,23). It is thought that hypermethylation of p16 silences the gene and increases c-myc expression, resulting in up-regulation of hTERT expression. If this is so, the elevation of telomerase activity observed in most cancer tissues and cancer cell lines is due to p16 methylation. In the present study, however, we investigated methylation not only of hTERT but also of p16 and we showed that up-regulation of hTERT expression does not originate from hypermethylation of p16 because the frequency of p16 hypermethylation was lower than that of up-regulation of hTERT. The Myc oncogene activates hTERT expression and c-myc works as a transcription factor in conjunction with Max protein and the Mad family proteins (23–25). Therefore, the Myc/Max/Mad network affects hTERT expression and methylation of the genes encoding these proteins might affect hTERT expression. Recently, it was reported that progesterone regulates hTERT expression via activation of the mitogen-activated protein kinase signaling pathway (26). Methylation status of the genes in this signaling pathway may also affect hTERT expression. Additional genes that are silenced by hypermethylation may cause down-regulation of hTERT expression. To clarify this point, further studies are needed.
In normal mucosa of six cases, hTERT expression was weakly observed. BiPS analysis revealed partial methylation of hTERT gene. This may be due to the presence of cancer cells present in normal mucosa or to mononuclear cells or immature cells expressing hTERT. Some telomerase activity is in peripheral blood cells (27) that originate from a subset of somatic cells such as lymphocytes, intestinal mucosal cells and skin basal cells.
We compared the methylation status of the promoter region of the hTERT gene with expression of other methylated genes (HIC1, p16, hMLH1) (15). The hypermethylation of hTERT gene promoter was found most frequently among the examined genes and did not correlate well with the methylation status of other genes. It suggests that silence of the hTERT expression is more critical in carcinogenesis than that of the other genes.
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Acknowledgments |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
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This work was supported in part by Grants-in-Aid for Cancer Research and for the Second Term Comprehensive 10-Year Strategy for Cancer Control from the Ministry of Health and Welfare, Japan, by the Foundation for Promotion of Cancer Research in Japan and by a Grant-in-Aid from the Vehicle Racing Commemorative Foundation.
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FOOTNOTES |
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+ For reprints and all correspondence: Masato Maekawa, Department of Laboratory Medicine, Hamamatsu University School of Medicine, Handayama 1–20–1, Hamamatsu, 431-3192, Japan. E-mail: mmaekawa@hama-med.ac.jp
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Received June 12, 2001; accepted October 3, 2001.
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