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Molecular Biology, Pathobiology and Genetics |
1 Department of Molecular Cytogenetics, Medical Research Institute and Graduate School of Biomedical Science and 2 Center of Excellence Program for Frontier Research on Molecular Destruction and Reconstitution of Tooth and Bone, Tokyo Medical and Dental University; 3 Cancer Genomics Project, National Cancer Center Research Institute, Tokyo, Japan; 4 Core Research for Evolutionary Science and Technology of the Japan Science and Technology Corporation, Saitama, Japan; and 5 Department of Pediatrics, Kyoto Prefectural University of Medicine, Kyoto, Japan
Requests for reprints: Johji Inazawa, Department of Molecular Cytogenetics, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, 113-8510 Tokyo, Japan. Phone: 81-3-5803-5820; Fax: 81-3-5803-0244; E-mail: johinaz.cgen{at}mri.tmd.ac.jp.
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Abstract |
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Introduction |
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Because hypermethylation in CpG-rich promoter or exonic regions seems to be a critical contributor to inactivation of tumor suppressor genes in many human cancers through transcriptional silencing (9), identification of hypermethylated CpG-rich sequence in cancer cell genomes could accelerate identification of unknown tumor suppressors. Although several techniques, including a method known as methylated CpG island amplification (MCA), have been developed (10, 11), we still have limited number of effective and practical high-throughput methods for genome-wide screening of aberrantly methylated CpG-rich sequences. To accomplish high-throughput screening for methylated sites in the entire genome, we developed a bacterial artificial chromosome (BAC) array-based MCA (BAMCA), incorporating our custom-made, BAC-based genomic DNA array combined with MCA (12).
In an effort to identify genes that are silenced by methylation mechanisms and associated with progression of neuroblastoma, we applied BAMCA to human neuroblastoma in the study reported here. Because the pattern of genomic changes observed in most neuroblastoma-derived cell lines is similar to that of advanced primary neuroblastomas (13), we used DNAs from neuroblastoma cell lines and from stage I primary tumors as test and reference samples, respectively. Using this approach, we successfully identified one gene, nuclear receptor 1I2 (NR1I2), also known as PXR, whose expression was decreased in a subset of human cell lines and tumors of neuroblastoma through hypermethylation of a CpG island showing promoter activity. NR1I2 was methylated and silenced mainly in late-stage neuroblastoma tumors with MYCN amplification and in older children. Exogenous restoration of NR1I2 expression suppressed growth of neuroblastoma cells lacking endogenous expression of the gene.
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Materials and Methods |
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Primary tumor samples were obtained at surgery from 51 neuroblastoma patients who underwent tumor resection at University Hospital, Kyoto Prefectural University of Medicine from 1986 to 2003, with written consent from the parents of each patient in the formal style and after approval by the local ethics committees. Staging was evaluated according to the criteria of the International Neuroblastoma Staging System (14). Of the 51 cases, 12 were classified as stage I, 11 as stage II, eight as stage III, 13 as stage IV, and four as stage IVS. Thirty-seven of the patients were infants <1 year of age at diagnosis. MYCN amplification was detected in 8 of 51 cases (15%). In 39 cases (76%), neuroblastoma had been detected by a mass screening program. Patients were treated according to previously described protocols (15, 16). Tumor samples were frozen immediately and stored at –80°C until required.
Bacterial artificial chromosome array-based methylated CpG island amplification. The preparation of DNA probes for screening of methylated regions was carried out by the MCA method described by Toyota et al. (11). Five-microgram aliquots of test DNA were first digested with 100 units of a methylation-sensitive restriction enzyme SmaI and subsequently with 20 units of methylation-insensitive XmaI. Adaptors were ligated to XmaI-digested sticky ends and PCRs were done with an adaptor primer and Cy3-dCTP for labeling. Control DNA was treated in the same manner except that labeling was with Cy5-dCTP (Fig. 1A).
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Reverse transcription-PCR and real-time quantitative reverse transcription-PCR. Single-stranded cDNAs were generated from total RNAs (17) and amplified with specific primers for each gene. Primer sequences are available on request. Real-time quantitative PCR was done using LightCycler (Roche Diagnostics, Tokyo, Japan) with SYBR green as described previously (18). The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene served as an endogenous control. Each sample was normalized on the basis of its GAPDH content. PCR amplification was done in duplicate for each sample.
Methylation analysis. To investigate methylation of DNA, the method of combined bisulfite restriction analysis (COBRA) was done as described earlier (11). Genomic DNAs were treated with sodium bisulfite and subjected to PCR using primer sets designed to amplify the regions of interest. PCR products were digested with HhaI, which recognizes sequences unique to the methylated alleles but cannot recognize unmethylated alleles, and electrophoresed. For bisulfite sequencing, PCR products were subcloned and sequenced.
Reporter assay. A 1,060 bp fragment upstream of exon 1 of NR1I2 (region 1; Fig. 1A) and a 480 bp fragment of a CpG island that includes exon 3 (region 2; Fig. 1A) were ligated into the pGL3-Basic vector (Promega, Madison, WI) in front of and/or downstream of the luciferase gene. An equal amount of each construct was introduced into cells with an internal control vector (pRL-hTK, Promega), using FuGENE 6 (Roche Diagnostics). A pGL3-Basic vector without insert served as a negative control. Firefly luciferase and Renilla luciferase activities were each measured 36 hours after transfection using the Dual-Luciferase Reporter Assay System (Promega); relative luciferase activities were calculated and normalized versus Renilla luciferase activity.
Transfection, Western blotting, and colony formation assays. A full-length NR1I2 cDNA was cloned into the pCMV-Tag3 eukaryotic expression vector (Stratagene, La Jolla, CA) with or without etoposide (VP-16) in-frame along with the Myc epitope. A plasmid expressing a Myc-tagged NR1I2 with or without VP-16 (pCMV-Tag3-VP-NR1I2 or pCMV-Tag3-NR1I2), or the empty vector (pCMV-Tag3-mock), were transfected into cells using FuGENE6 (Roche Diagnostics). Expression of NR1I2 protein in transfected cells was confirmed by Western blotting using anti-Myc-Tag antibody (9B11; Cell Signaling Technology, Beverly, MA). For colony formation assays, transfected cells were selected with 500 µg/mL G418; 3 weeks after transfection, the neomycin-resistant colonies were stained with crystal violet and counted (17).
Cell growth assay. Stable NR1I2 transfectants and controls were obtained by transfecting pCMV-Tag3-VP-NR1I2 or pCMV-Tag3-mock, respectively, into cells lacking NR1I2 expression. For measurements of cell growth, 2 x 103 cells were seeded in 96-well plates. The numbers of viable cells were assessed by a colorimetric water-soluble tetrazolium salt assay (cell counting kit-8; Dojindo Laboratories, Kumamoto, Japan).
Oligonucleotide array analysis. mRNA expression profiling was done using the AceGene Human oligo chip 30K (DNA Chip Research, Inc., Kanagawa, Japan), containing 30,000 genes, as described elsewhere (18). The test and reference cDNA probes labeled with aminoallyl-dUTP (Ambion, Inc., Austin, TX) were synthesized using oligo(dT)12-18 primer and coupled with Cy3- or Cy5-monoreactive dye (Amersham Biosciences, Tokyo, Japan), respectively. The hybridized chips were scanned using GenePix 4000B (Axon Instruments) and analyzed using GenePix Pro 4.1 software (Axon Instruments). Signal intensities between the two fluorescent images were normalized by the averaged values for blank spots; this procedure effectively defined the signal intensity-weighted spot for the internal controls of housekeeping genes on each array to have a Cy3/Cy5 ratio of 1.0.
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Results |
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To investigate whether demethylation could restore NR1I2 mRNA in neuroblastoma cells lacking endogenous expression, we treated cells with 1 µmol/L of 5-aza-dCyd, a methyltransferase inhibitor, for 5 days. Expression of NR1I2 mRNA was remarkably increased after the treatment (Fig. 1D).
Methylation of NR1I2 CpG island in neuroblastoma cell lines. We next examined the methylation status of the slightly CpG-rich 5' region (region 1) and the CpG island including exon 3 (region 2) of the NR1I2 gene, which had been detected by the National Center for Biotechnology Information (NCBI) human genome database6 as shown in Fig. 1E. Bisulfite sequencing analysis of region 2 revealed aberrant DNA hypermethylation in IMR32 and SH-SY5Y cell lines lacking expression of NR1I2, but hypomethylation in two lines expressing the gene (SK-N-AS and SK-N-KP) and in a normal lymphoblast cell line (LCL). On the other hand, no significant difference in methylation pattern within region 1 was observed among those four neuroblastoma cell lines and LCL, regardless of expression status. We did COBRA to confirm the relationship between expression and methylation status within region 2 in a larger set of neuroblastoma cell lines. Predominant methylated alleles were detected in all lines lacking NR1I2 expression (Fig. 1F and data not shown).
Because several splicing variants of NR1I2, including the variant starting transcription from exon 3 (variant 4, v4), have been reported in various human tissues (19),7 we did RT-PCR using specific primers for each variant to examine which transcripts might be silenced through a DNA methylation within region 2 in neuroblastoma cell lines (Fig. 1G). Variant 1, the most major variant in various human tissues, and variant 2 were expressed in unmethylated neuroblastoma cell lines, but not in methylated lines, whereas variant 4 without open reading frame was not expressed in one of the unmethylated cell lines (SK-N-AS). The expression of variant 1 and variant 4 was restored after the treatment with 5-aza-dCyd in methylated neuroblastoma cell lines, whereas the expression of variant 2 was not. Variant 3, lacking a part of exon 5, was not expressed in neuroblastoma cell lines regardless of methylation status within CpG island and 5-aza-dCyd treatment. Those results suggested that the methylation of CpG residues in region 2 might be mainly responsible for the silencing of variant 1 of the NR1I2 gene starting transcription from exon 1a in neuroblastoma, although region 2 does not contain its transcriptional start site.
Promoter activity of the CpG island located around exon 3 of NR1I2. Because the CpG island (region 2) of NR1I2 was located around exon 3, we first determined whether region 2 had promoter activity by means of a luciferase reporter assay. This fragment alone (Fig. 2A, 2/L) revealed clear promoter activity, whereas region 1 fragment upstream of exon 1 (Fig. 2A, 1/L) showed almost none (Fig. 2B). In addition, we next determined whether region 2 acts as an enhancer to stimulate transcription from exon 1 by testing the luciferase activity in construct containing region 1 in front of the luciferase reporter gene and region 2 downstream of the luciferase gene (Fig. 2A, 1/L/2). Although region 2 downstream of the luciferase gene (Fig. 2A, L/2) showed some promoter activity, region 2 revealed no enhancer activity for region 1 (Fig. 2C).
Analysis of NR1I2 methylation and expression in primary neuroblastoma tumors. We next examined methylation status of the NR1I2 CpG island in 51 surgically resected primary neuroblastomas using COBRA (Table 2). Clearly methylated alleles were detected in nine of the tumors (17%; Fig. 3A). The appearance of partial methylation observed in those nine tumors can be explained by the unavoidable contamination of nontumorous cells in the specimens. Five of the nine tumors (55%) had undergone MYCN amplification, whereas only 3 of 42 (7%) unmethylated tumors showed amplification of MYCN (Table 2). Moreover, methylation of region 2 of the NR1I2 gene was more frequently detected in advanced tumors (stages III and IVa; P = 0.0234, Fisher's exact test), tumors from patients with poor outcome (dead from disease; P = 0.0135, Fisher's exact test), and tumors from patients >1 year old (P = 0.052, Fisher's exact test), although the difference did not quite reach statistical significance in terms of patient age. In the 47 neuroblastoma cases where high-quality RNAs were available for expression analysis, a clear correlation between the methylation status of the CpG island and expression level of NR1I2 mRNA was observed (Fig. 3B and C). By means of real-time quantitative RT-PCR experiments, we saw a statistically significant inverse correlation between expression of NR1I2 mRNA and tumor stage (P = 0.0137, Mann-Whitney U test) or MYCN amplification (P = 0.0003, Mann-Whitney U test; Fig. 3C).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BAMCA, however, has some disadvantages: (a) It examines only a limited number of CpG sites within a CpG island because only SmaI/XmaI sites are used to search differentially methylated CpGs. (b) It identifies BACs, which contain differentially methylated sequences between test and reference DNAs, although sequences are not always associated with promoter regions of genes. (c) It misses differentially methylated sequences/genes without BACs spotted on the BAC array used. To improve the sensitivity of array-based methylation screening, it is possible to use other methylation-sensitive restriction sites as indicators, including NotI site (20). However, BAMCA may quickly provide the list of possible target genes for methylation within identified BAC clones using information from human genome database without cloning and sequencing of enriched fragments, suggesting that it may have important applications in population-based studies of CpG island methylation.
NR1I2 locates at 3q13.33, a chromosomal region that is not often involved in loss of heterozygosity or copy number losses in neuroblastoma (21, 22). Indeed, most of the cell lines we used in this study showed normal copy number in 3q (13), suggesting that a homozygous inactivation of NR1I2 might occur by biallelic methylation. Similar findings have been reported for several genes, such as RASSF1 (3p21.3), DAPK (9q34.1), and THBS1 (15q15), which are located in regions not frequently deleted, although some methylated genes, such as ARF and INK4A (9p21), and CASP8 (2q23), are in fact on regions frequently deleted in neuroblastoma (23). Therefore, both biallelic methylation and monoallelic methylation with allele loss may be important mechanisms for inactivating tumor-associated genes in this disease.
Our promoter assays showed that CpG island around exon 3 (region 2) shows promoter activity, but CpG-rich 5' region containing exon 1 and its 5' upstream sequences (region 1) does not. Region 2 shows no enhancer activity for region 1 (Fig. 2). The methylation status of region 2, but not region 1, was highly and inversely correlated with the expression of NR1I2, especially the most major variant of NR1I2, variant 1, starting from exon 1a. Those results suggest that the methylation status of CpG residues in region 2 might be responsible for the silencing of this gene and contributed to loss of function of NR1I2 protein in neuroblastoma. A few studies, including ours, have shown that promoter activity can occur in fragments, especially CpG islands, not containing transcriptional starting sites (17, 24, 25). It is possible that methylation that occurred in those CpG islands with promoter activity may silence gene expression from specific starting sites.
Among numerous hypermethylated genes reported in neuroblastoma (3–8), CASP8 seems to be inactivated through promoter methylation in advanced neuroblastoma tumors where MYCN is amplified (4). Those findings, along with ours, suggest that (a) unknown mechanisms contribute to progression of neuroblastoma by causing genetic alterations, including MYCN amplification, as well as methylation-mediated inactivation of a subset of tumor suppressor genes; (b) methylation-mediated inactivation of a subset of tumor suppressor genes may cause genetic changes that lead to progression of neuroblastoma; or (c) MYCN amplification and/or other alterations in advanced neuroblastomas may bring about a CpG island methylator phenotype (3). Gonzalez-Gomez et al. (23) reported that higher aggressiveness, represented at the molecular level by concurrent MYCN amplification and 1p loss in neuroblastoma, was not paralleled by an accumulation of methylation events among various genes they examined, suggesting that CpG island methylation in advanced neuroblastoma could be specific to a subset of genes.
The NR1I2 gene encodes an orphan nuclear receptor that plays a key role in the regulation of xenobiotic response by controlling expression of drug metabolizing and clearance molecules (26–28). NR1I2 protein activates expression of genes encoding proteins such as CYP3A4 and ABCB1, which reduce the concentrations of xenochemicals and toxic bile acids (29). However, effects of NR1I2 on cell growth or expression of growth-regulating genes have never been clarified, although we have shown here that the induction of ectopic NR1I2 inhibited growth of neuroblastoma cells. Other nonsteroidal nuclear receptors, such as all-trans retinoic acid receptor and vitamin D3 receptor, which form heterodimers with the 9-cis retinoic acid receptor in the same way as NR1I2, mediate antiproliferative and differentiation-promoting activities toward several malignant cell types (30, 31). Therefore, growth-suppressive activity might be one of the normal functions of NR1I2 although its mechanisms remain unknown.
To achieve some clarity with respect to the growth inhibitory activity of NR1I2, we tried to determine its putative transcriptional targets. Among 105 genes through an expression array analysis, we selected 10 genes for validation by semiquantitative PCR based on their possible cancer-associated function (Online Mendelian Inheritance in Man),8 and identified one candidate, PLA2G2A, encodes secretory phospholipase A2, as a possible target of NR1I2, although it will be needed to determine whether PLA2G2A is a direct or indirect target. This product qualifies as a tumor suppressor because mice lacking PLA2G2A expression show increased colonic polyposis (32). Interestingly, PLA2G2A was mapped to chromosome 1p36, a region frequently implicated in the pathogenesis of neuroblastoma (33). Further screening of possible targets of NR1I2 will be necessary to clarify how NR1I2 regulates neuroblastoma cell growth.
Because only 7 of 51 neuroblastoma patients in our study died during follow-up periods, we did not perform a survival analysis. However, the high prevalence of NR1I2 silencing through DNA methylation that we observed in aggressive neuroblastomas, along with the shown growth suppression activity of NR1I2, indicate that this molecule might serve as a diagnostic marker to predict prognosis.
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Acknowledgments |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
We thank Ai Watanabe and Ayako Takahashi for technical assistance.
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Footnotes |
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Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).
6 http://www.ncbi.nlm.nih.gov/. 
7 http://www.ncbi.nlm.nih.gov/IEB/Research/Acembly/index.html.
8 http://www.ncbi.nlm.nih.gov/Omim/omimhelp.html.
Received 3/30/05. Revised 9/ 2/05. Accepted 9/13/05.
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, unmethylated CpG sites; 
, methylated CpG sites, respectively; each row represents a single clone. Arrows, HhaI restriction site. Arrowheads, SmaI restriction site. F, representative results of COBRA of region 2 in neuroblastoma cell lines with (+) or without (–) NR1I2 expression. A 492 bp PCR product, including exon 3, was restricted by HhaI. M, methylated alleles. G, top, map of four variants (v1-v4) and location of each primer set used for RT-PCR analysis. Black boxes, coding exons; gray box, deleted region in variant 3. Arrows, primers used for RT-PCR shown in (B and C), and in 
