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Keywords:

  • hypermethylation;
  • EDNRB;
  • nasopharyngeal carcinoma;
  • chromosome 13q;
  • tumor suppressor

Abstract

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

To identify the epigenetic changes in nasopharyngeal carcinoma (NPC), we performed methylation-sensitive restriction fingerprinting (MSRF) analysis on NPC cell lines and xenografts. A 190 bp sequence methylated in NPC tumors was isolated and showed high homology to the 5′ CpG island of the endothelin receptor B (EDNRB) gene. Since the EDNRB gene is commonly inactivated in prostate and bladder cancers, it may be a candidate target gene involved in NPC tumorigenesis. By bisulfite sequencing, we have confirmed that hypermethylation of the 5′ CpG island of EDNRB occurred in both xenografts and all 4 cell lines but not in 2 normal nasopharyngeal outgrowths. RT-PCR demonstrated that only original EDNRB transcripts, but not the splicing transcripts, were expressed in normal nasopharyngeal epithelial cells. Loss of the original EDNRB expression was consistently found in 2 xenografts and 3 cell lines with dense methylation patterns. Treatment of these 3 cell lines with 5′-aza-2′-deoxycytidine led to re-expression of the EDNRB transcript and demethylation of its promoter regions. Our results demonstrate that silencing of EDNRB gene expression in NPC is associated with promoter hypermethylation. Using methylation-specific PCR, we also detected methylation of the 5′ CpG island of EDNRB in 19/21 (90.5%) primary tumors, while no methylation was found in all 6 normal nasopharyngeal epithelia. The high frequencies of promoter hypermethylation suggest that repression of the EDNRB gene may play a role in the development of NPC. © 2002 Wiley-Liss, Inc.

Nasopharyngeal carcinoma (NPC) is a malignant disease prevalent in southern China. One unique feature of this cancer is its close association with latent Epstein-Barr virus (EBV) infection. The presence of clonal EBV genome and latent viral protein was demonstrated in almost all undifferentiated NPCs.1 Our previous genomewide studies, by comparative genomic hybridization (CGH) and allelotyping, revealed high frequencies of deletion on multiple chromosomal regions, including 3p, 9p, 11q, 13q, 14q and 16q.2, 3 Although several common deletion regions were delineated in our previous studies, only a limited number of tumor-suppressor genes on these regions have been identified as targets for inactivation in NPC.2–8 These include the RASSF1A and p16 genes on chromosomes 3p21.3 and 9p21, respectively.9–11 Aberrant methylation of the 5′ CpG island is the major mechanism for inactivation of these genes. Therefore, identification of methylated CpG sequences in the NPC genome could lead to the discovery of a cancer-related gene. In our pilot study, we searched for aberrantly methylated sequences by methylation-sensitive restriction fingerprinting (MSRF) analysis on several established NPC cell lines, xenografts and normal nasopharyngeal epithelium outgrowths.12 We identified one of these sequences that showed high homology to the 5′ CpG island of the endothelin receptor B (EDNRB) gene. The gene was mapped to chromosome 13q22, a region commonly deleted in NPC.2, 8 Endothelin receptor B mediates the mitogenic effect of the ET-1 pathway. Decreased expression and promoter hypermethylation of the EDNRB gene have been demonstrated in prostate cancer, bladder cancer, colorectal cancer and melanoma.13–17 Thus, EDNRB may be a candidate target for inactivation during the development of NPC. In the present study, we investigated whether promoter methylation of the EDNRB gene might be involved in the tumorigenesis of NPC. The methylation status of the EDNRB gene in the NPC samples, including 4 cell lines, 2 xenografts and 21 primary tumors, was explored.

MATERIAL AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Cell lines and tissue samples

Four NPC cell lines (C666-1, HK-1, CNE-1 and CNE-2) and 2 NPC xenografts (xeno-2117 and xeno-1915) were studied. Two of the lines (HK-1 and CNE-2) were derived from differentiated cancers, whereas the others were from undifferentiated cancers. Two normal epithelial cell outgrowths (NP-1 and NP-2), derived from nasopharyngeal mucosa, were examined. We also studied 21 primary tumors from NPC patients with consent before treatment at the Department of Clinical Oncology, Prince of Wales Hospital, Chinese University of Hong Kong. All of the NPCs were undifferentiated carcinomas according to the WHO classification.18 Tumor samples were histologically examined and shown to contain at least 70% tumor cells. Microdissection was performed manually to obtain a pure and enriched population of tumor cells from the samples with contamination of nonneoplastic cells. Genomic DNA was prepared from tissue samples and cell lines according to conventional methods.4

MSRF

Genomic DNA samples from the 4 NPC cell lines, 2 xenografts and 2 normal nasopharyngeal epithelial cell outgrowths were subjected to MSRF analysis as described.12 DNA (1 μg) was digested with 20 units MseI alone or 20 units each of BstUI and MseI. MseI-digested and BstUI/MseI-digested DNA were amplified with primers Bs12 (5′-GCCCCCGCGA-3′) and BS13 (5′-CGGGGCGCGA-3′). Thirty cycles of amplification (94°C for 2 min, 40°C for 1 min and 72°C for 2 min) were performed for each sample. PCR products were size-fractionated on a 5% nondenaturing polyacrylamide gel. After electrophoresis, gels were dried and autoradiographed for 1 to 2 days. Aberrantly methylated bands on the gel were eluted, reamplified and cloned using the pMOSBlue blunt-ended cloning kit (Amersham, Aylesbury, UK). Cloned aberrant DNA fragments were purified and then sequenced by the ABI PRISM DyeDeoxy Terminator Cycle Sequencing Kit and the ABI 377 DNA Sequencer (Applied Biosystems, Foster City, CA). The resulting sequence of the DNA fragments was subjected to the BLAST program (NCBI) for homology search.

Methylation analysis

Genomic DNA (1 μg) was subjected to bisulfite modification by the CpGenomic DNA Modification Kit (Intergen, Purchase, NY) according to the manufacturer's recommendations. The methylation status of 94 CpG dinucleotides in the region 5′' upstream of the EDNRB gene was examined in NPC cell lines and xenografts by bisulfite sequencing. A 1,608 bp sequence including the promoter region and exon 1 of the EDNRB gene was amplified by 3 primer pairs [E1F 5′-TGGTG(C/T)GTGATAATTTGTTTTT-3′ and E1R 5′-AACCC(G/A)CAAAAACTTCTCAA-3′, E2F 5′-TAGGTTGGGTAAAGGAAGGA-3′ and E2R 5′-ACTACATACTACTACCTACTCCAAAA-3′, E3F 5′-TTGGAGTAGGTAGTAGTATGTAGT-3′ and E3R 5′-TCCC(G/A)ATAATCCCCAACAC-3′]. Amplified fragments were subcloned using the pMOSBlue blunt-ended cloning kit. Five clones of each sample were sequenced by the ABI PRISM DyeDeoxy Terminator Cycle Sequencing Kit and the ABI 377 DNA sequencer. The promoter methylation status of the EDNRB gene in the 21 primary NPCs was investigated by methylation-specific PCR (MS-PCR) as described.19 Bisulfite-modified DNA was amplified with either a methylation-specific or a nonmethylation-specific primer set at 35 cycles: 95°C for 30 sec, 58°C for 30 sec and 72°C for 30 sec. Two regions within the 5′ CpG island of EDNRB were examined (Fig. 2). The first set of primers spanned 5 CpG sites at the promoter region. Primers for methylated DNA were R1M-F (5′-GAGCGCGAGTGTTTGAAGTC-3′) and R1M-R (5′-AAACTACAACTCCCGCAACG-3′), while those for unmethylated DNA were R1U-F (5′-TTGGGTAAAGGAAGGAGTGTG-3′) and R1U-R (5′-CCACAACACACCCAAAAATACA -3′). The other primer sets were designed for examining the methylation status of 6 CpG sites within exon 1. Primers for methylated DNA were R2M-F (5′-AAATTGCGGAGCGGTTATC-3′) and R2M-R (5-CCAAATCCGCGACAAACCG-3′). Primers for unmethylated DNA were R2U-F (5′-GGTTTTGAAATTGTGGAGTGG-3′) and R2U-R (5′-TCCCCAAATCCACAACAAAC-3′). Positive control (C666-1) were performed for each set of reactions.

Treatment with 5′-aza-2′-deoxycytidine

Three cell lines, C666-1, HK-1 and CNE-2, were treated with varying concentrations of 5′-aza-2′-deoxycytidine (Sigma, St. Louis, MO), ranging from 1 to 10 μM, the day after plating. Culture medium and drug were replaced every 24 hr. After 5 days' exposure to 5′-aza-2′-deoxycytidine, cells were harvested for DNA and RNA extraction.

RT-PCR

RT-PCR was performed in a normal nasopharyngeal epithelial outgrowth (NP-1), NPC cell lines and xenografts. Total RNAs were extracted from NPC samples using Trizol reagent (Life Technologies, Rockville, MD) according to the manufacturer's recommendations. Primers and PCR conditions for 4 different transcripts (EDNRBΔ1, EDNRBΔ2, EDNRBΔ3 and original EDNRB) were described previously.20 RNA samples were also amplified by the primers of the CDK4 gene as control.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Identification of methylated sequences in NPC

By MSRF, 3 abnormally methylated sequences were detected in NPC tumors using a pair of arbitrary primers. Only one of them showed hypermethylation in all NPC samples (Fig. 1a). The aberrantly methylated DNA fragment Bs12/13-1 was isolated and sequenced. The sequence of the 190 bp fragment is shown in Figure 1b. By the BLAST program, this isolated fragment showed high homology to nt 585 to 768 of the region 5′ upstream of the EDNRB gene (accession number D13162, GI 285918). Identification of the aberrantly methylated fragment in the 5′ CpG island of the EDNRB gene by MSRF analysis suggested that hypermethylation of this gene is associated with the development of NPC.

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Figure 1. (a) MSRF analysis of NPC. Patterns of PCR products amplified from MseI-digested and BstU1/MseI-digested samples including 2 normal NPC epithelial outgrowths (NP-1, NP-2), 2 NPC xenografts (xeno-2117, xeno-1915) and 1 NPC cell line (HK-1). Arrow shows the hypermethylated DNA fragment Bs12/13-1 in NPC samples. (b) Nucleotide sequences of the methylated fragment Bs12/13-1. Boxed region shows the sequence of the primers used in MSRF analysis.

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Methylation of the EDNRB gene in NPC

To investigate the methylation status of the 5′ CpG islands of the EDNRB gene in NPC, we performed bisulfite sequencing on 2 normal nasopharyngeal outgrowths, 2 NPC xenografts and 4 cell lines. Since the EDNRB gene contains a large CpG island, 94 CpG dinucleotides spanning 1608 bp of the 5′ upstream region were investigated by bisulfite sequencing. The sequence examined include the promoter region and exon 1 of the gene. Methylation patterns of the EDNRB gene in the NPC samples are shown in Figure 2a. We detected methylation of multiple CpG sites in all xenografts and cell lines but not in 2 normal nasopharyngeal outgrowths. Dense methylation of the 94 CpG sites was observed in xeno-2117, xeno-1915, C666-1, HK-1 and CNE-2; almost all CpG sites were highly methylated. Complete methylation of the CpG sites flanking the transcription start site of the original EBNRB transcripts was detected in these samples. In CNE-1, most of the CpG sites showed partial or no methylation. Only few CpG sites were partially methylated in 2 normal nasopharyngeal outgrowths. Furthermore, we analyzed the methylation status of the EDNRB promoter in 21 primary tumors by MS-PCR. Figure 2b shows MS-PCR analysis of the EDNRB promoter in the NPC samples. Two distinct regions of the 5′ CpG island of EDNRB were examined. Of the 21 primary tumors, 19 (90.5%) were methylated in both regions analyzed by MS-PCR. Six microdissected normal nasopharyngeal epithelia showed no methylation of the EDNRB promoter. Thus, a high frequency of promoter hypermethylation of the EDNRB gene in NPC was demonstrated.

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Figure 2. (a) Bisulfite sequencing results of the 5′ upstream region of EDNRB in normal nasopharyngeal epithelial outgrowths (NP-1, NP-2), NPC cell lines (C666-1, HK-1, CNE-2, CNE-1) and xenografts (xeno-2117, xeno-1915). Each circle indicates a CpG dinucleotide. Five clones from each sample were analyzed. The percentage of 5-methylcytosine for 94 CpG sites of each sample is shown. Black arrows indicate primer pairs for bisulfite sequencing. The positions of the Bs12/13-1 sequence and the regions for MS-PCR analysis (regions 1 and 2) are indicated. (b) MS-PCR analysis of NPC primary tumors and normal nasopharyngeal epithelia. The methylation status of the samples was analyzed using specific primers for 2 distinct regions (regions 1 and 2) of the 5′ CpG island of EDNRB. Reactions specific for methylated DNA (m) or for unmethylated DNA (u) are indicated.

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Expression of EDNRB in NPC

Expression of the EDNRB transcripts in a normal nasopharyngeal cell outgrowth, 2 NPC xenografts and 4 NPC cell lines was examined by RT-PCR. Four different transcripts (EDNRBΔ1, EDNRBΔ2, EDNRBΔ3 and original EDNRB) were investigated. We found no expression of EDNRBΔ1, EDNRBΔ2 and EDNRBΔ3 in all NPC xenografts, cell lines and normal nasopharyngeal outgrowths. However, the original EDNRB transcripts were detected in normal nasopharyngeal outgrowths and 1 NPC cell line (CNE-1), showing partial methylation of the EDNRB promoter (Fig. 3a). No expression of normal transcripts was detected in the NPC cell lines (C666-1, HK-1 and CNE-2) and xenografts (xeno-2117 and xeno-1915) with dense methylation. Our results demonstrated that only the original EDNRB transcripts were expressed in nasopharyngeal epithelial cells. Furthermore, expression of these transcripts was silenced in the tumors with promoter hypermethylation.

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Figure 3. (a) Original EDNRB transcript expression in normal and malignant nasopharyngeal samples. RT-PCR was carried out to determine the original EDNRB transcript expression in the normal nasopharyngeal epithelial outgrowth (NP-1), NPC cell lines (C666-1, HK-1, CNE-1, CNE-2) and xenografts (xeno-2117, xeno-1915). Transcripts were detected in NP-1 and CNE-1 only. PCR products in CNE-1 are indicated with a white arrow. Expression of CDK4 in these samples was used as a control. (b) Restoration of original EDNRB transcript expression in NPC cell lines after 5′-aza-2′-deoxycytidine treatment. Three NPC cell lines (C666-1, HK-1, CNE-2) were treated for 4 days with 0, 1 and 3 μM 5′-aza-2′-deoxycytidine. Re-expression of EDNRB transcripts was detected in all 5′-aza-2′-deoxycytidine–treated cell lines. (c) The methylation status of the 5′-aza-2′-deoxycytidine–treated NPC cell line C666-1. MS-PCR analysis of regions 1 and 2 of the 5′ CpG island of EDNRB demonstrated that the unmethylated sequences were detected in samples treated with 5′-aza-2′-deoxycytidine (1 or 3 μM) but not in untreated cells.

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Re-expression of EDNRB in NPC cell lines

To investigate whether suppression of EDNRB gene expression was mediated by promoter hypermethylation in NPC cells, 3 cell lines (C666-1, HK-1, and CNE-2) were treated with 5′-aza-2′-deoxycytidine for 4 days at various concentrations. Gene expression was restored in all 3 NPC cell lines after treatment (Fig. 3b), and the EDNRB promoter of these cell lines was partially demethylated, as analyzed by MS-PCR (Fig. 3c). These results suggested that methylation of the promoter region may play a functional role in silencing the expression of EDNRB in NPC.

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Epigenetic changes are common in human cancers and play an important role in the development of cancer.21, 22 Transcriptional silencing by promoter hypermethylation is becoming recognized as a common mechanism for inactivation of tumor-suppressor genes. Genomewide searches for hypermethylated sequences may facilitate the identification of aberrantly methylated 5′ CpG islands in tumor samples and the isolation of novel or known cancer-related genes whose expression is repressed by methylation. In the present study, we identified a sequence consistently methylated in NPC samples by MSRF analysis. The methylated sequence was further confirmed to be part of the 5′ CpG island of the EDNRB gene. The gene encodes a nonselective endothelin B receptor (ETB). ETB mediates endothelin-1 expression and may be associated with cancer proliferation. Decreased ETB expression was found in advanced prostate cancer in vitro and in vivo.14 However, the role of the EDNRB gene in tumorigenesis remains unclear. In our previous microsatellite analysis, we also detected a high frequency of allelic loss on chromosome 13q, in which the EDNRB gene is located.2, 8 Deletion of the region containing the EDNRB gene was found in about 50% of NPC samples.2 Thus, EDNRB may be a candidate tumor-suppressor gene of NPC. Nelson et al.16 first detected methylation of the 5′ CpG island in 70% of prostate cancers by Southern blotting. Pao et al.17 reported promoter hypermethylation of the EDNRB gene in various human malignancies, including cancers of the prostate, bladder and colon. They also showed that methylation of the 5′ region of the gene was associated with expression of multiple EDNRB transcripts in these cancers. However, we have found only the original EDNRB transcripts in the normal nasopharyngeal epithelial cells and the cell line CNE-1 with partial methylation. Tsutsumi et al.20 demonstrated that the expression pattern of multiple transcripts varies between different tissues. Our findings revealed a distinct transcription pattern of EDNRB in nasopharyngeal epithelial cells. Comprehensive study of the 94 CpG sites in the 5′ region revealed dense methylation patterns in NPC xenografts (xeno-2117 and xeno-1915) and cell lines (C666-1, HK-1 and CNE-2) without EDNRB expression. Complete methylation of the EDNRB promoter has also been found in 4/5 prostate cancer cell lines while examining the CpG sites recognized by BSSHII.16 By Ms-SNuPE analysis, Pao et al.17 examined 11 CpG sites of the EDNRB promoter in human cancers, detecting dense methylation and loss of the EDNRB transcripts in 2/3 bladder cancer cell lines and 4/5 colon cancer cell lines. In the present study, we also observed a high frequency (90.5%) of promoter methylation in primary tumors while examining multiple CpG sites in the distinct regions of the 5′ CpG island by MS-PCR. No methylation of the EDNRB promoter in normal epithelial outgrowths and microdissected normal tissue suggested that the promoter methylation is tumor cell-specific. However, partial methylation has been found in normal colon tissue but not normal prostate and bladder samples.17 According to the hypothesis of Knudson,23 both alleles of a tumor-suppressor gene need to be inactivated for tumorigenesis. Although results of loss of heterozygosity studies on 13q or the EDNRB locus were not available, deletion or rearrangement of chromosome 13q is commonly found in NPC cell lines and xenografts by cytogenetic or CGH analysis.24–26 Since no unmethylated allele was found in almost all of these NPC samples, we suspected that loss of expression may be the result of complete inactivation of both alleles by promoter hypermethylation and allelic deletion in some of these tumors. Biallelic methylation is another possible mechanism for inactivation of EDNRB in NPC. We did not analyze loss of heterozygosity of 13q on these samples since no corresponding normal tissue was available. However, we previously detected loss of heterozygosity of this chromosomal region in about 56% of primary NPCs.2 We found promoter hypermethylation of this gene in 19/21 (90.5%) primary tumors. Taken together, these findings suggest that promoter hypermethylation and allelic loss may also be the major mechanisms for EDNRB inactivation in primary NPC. Treatment with the DNA methyltransferase inhibitor 5′-aza-2′-deoxycytidine led to re-expression of the original EDNRB transcripts and demethylation of the gene. Thus, promoter hypermethylation is directly responsible for transcriptional inactivation of the EDNRB gene in NPC cell lines. Correlation of promoter hypermethylation with loss of EDNRB transcripts was also demonstrated in the prostate cancer and bladder cancer cell lines.16, 17

In summary, we have detected a high incidence of aberrant methylation of the EDNRB gene in NPC. Promoter methylation was detected in 75%, 100% and 90.5% of the cell lines, xenografts and primary tumors, respectively. Our findings strongly suggest that epigenetic inactivation of the EDNRB gene may play a role in the tumorigenesis of NPC. Further transfection studies on the expression of wild-type EDNRB in NPC cell lines are ongoing, to investigate its biologic function in human cancers. The successful identification of the novel methylated target, EDNRB, sheds light not only on the genetic basis of NPC but also on the development of tumor markers. Tumor-specific methylation changes are promising tumor markers.27–29 For the EDNRB gene, promoter methylation was found in almost all NPC samples but not in normal nasopharyngeal tissue. This striking result suggests that such an epigenetic change may serve as a molecular marker for early detection and disease monitoring of NPC.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES