Human Molecular Genetics Advance Access originally published online on March 3, 2005
Human Molecular Genetics 2005 14(8):997-1007; doi:10.1093/hmg/ddi092
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Frequent silencing of DBC1 is by genetic or epigenetic mechanisms in non-small cell lung cancers
1Department of Molecular Cytogenetics, Medical Research Institute and School of Biomedical Science, 2Thoracic and Cardiovascular Surgery, 3Center of Excellence Program for Frontier Research on Molecular Destruction and Reconstitution of Tooth and Bone, Tokyo Medical and Dental University, Tokyo 113-8510, Japan, 4Core Research for Evolutional Science and Technology of Japan Science and Technology Corporation, Saitama 332-0012, Japan, 5Tokyo Kyosai Hospital, Tokyo 153-8934, Japan and 6Pathology Division, National Cancer Center Research Institute, Tokyo 104-0045, Japan
* To whom correspondence should be addressed at: Department of Molecular Cytogenetics, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan. Tel: +3 58035820; Fax: +3 58030244; Email: johinaz.cgen{at}mri.tmd.ac.jp
Received December 18, 2004; Revised February 15, 2005; Accepted February 22, 2005
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Genome-wide screening of DNA copy number aberrations in 27 cell lines derived from non-small cell lung cancers (NSCLCs), using a custom-made comparative genomic hybridization (CGH)-array, identified a homozygous deletion of the deleted in bladder cancer 1 gene (DBC1) in one cell line. Homozygous deletion of DBC1, located at 9q33.1, was also observed in two of 53 primary NSCLC tumors examined. Moreover, 21 of the other 26 cell lines showed complete loss of DBC1 expression, although normal lung tissues express this gene, and treatment with 5-aza-2'-deoxycytidine restored expression of DBC1. Hypermethylation in part of a CpG island around the exon 1 of DBC1 has been reported in urothelial cancers, but the potential association between methylation and expression status was never clarified in that disease. In our experiments, a different part of the same CpG island showed promoter activity in vitro and was frequently methylated in our cell lines and primary tumors of NSCLC, where methylation status correlated inversely with gene expression. Among our primary NSCLC cases, methylation of the DBC1 promoter occurred more frequently in men, elderly patients and smokers than in women, younger patients and nonsmokers respectively, but it was not correlated with tumor stage or histology. Exogenous overexpression of DBC1 in NSCLC cell lines lacking its expression inhibited cell growth. Our results provide the first evidence that DBC1 is a likely tumor suppressor for NSCLC; silencing of the gene through homozygous deletion or methylation of its promoter region may be associated with progression of this disease.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Lung cancer is currently the leading cause of cancer mortality in the United States as well as Japan, and its incidence is still increasing (1
,2). Non-small cell lung cancer (NSCLC) accounts for 80% of all cases, and prognosis is generally unfavorable for patients with this type of lung tumor. Since multiple genetic alterations occurring sequentially in a cell lineage underlie the carcinogenetic process in NSCLC, identification of specific alterations could suggest optimal molecular targets for clinical management of this intractable type of cancer. Many genetic alterations remain to be identified, suggesting that high-resolution screening of the entire genome could reveal critical alterations that have been missed by the methods applied to date.
Comparative genomic hybridization (CGH) using arrayed genomic DNAs, such as those from bacterial artificial chromosome (BAC)/P1-artificial chromosome (PAC) libraries, is currently the most powerful method to detect and simultaneously localize losses or gains of genetic material throughout the genome. Combined with the human genome database, CGH arrays enable high-throughput quantitative measurement of DNA copy number at high resolution, allowing rapid discovery of novel tumor suppressor genes as well as oncogenes in cancer genomes (3–6).
In the work reported here, we refined the genetic characterization of a panel of 27 NSCLC cell lines using our custom-made BAC/PAC-based CGH array (5,6), and identified homozygous loss of the deleted in bladder cancer 1 gene (DBC1, located at 9q33.1) in one cell line. Homozygous loss and hypermethylation of DBC1 had been described in some urothelial tumors (7–9). As loss of heterozygosity (LOH) at 9q is relatively frequent in NSCLCs (10–12), we focused on DBC1 as a possible target of the mechanism(s) leading to LOH. Although correlation between expression and the methylation status of DBC1 had remained unclear in urothelial tumors (9), expression of this gene was frequently silenced in our panel of NSCLC cells without homozygous loss. Silencing of the gene in those cells had occurred through hypermethylation of a region within a CpG island exhibiting promoter activity. We also observed homozygous deletion of DBC1 and hypermethylation of its CpG island in some primary NSCLCs. Moreover, exogenous expression of DBC1 in DBC1-nonexpressing NSCLC-derived cells suppressed their growth. Our results suggested that DBC1, which appears to be inactivated frequently in NSCLCs through genetic and/or epigenetic mechanisms, may function as a tumor suppressor in human lung.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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CGH array analysis of NSCLC cell lines
High-throughput identification of homozygous deletions in cell genomes is a powerful way to identify candidate tumor suppressor genes that are susceptible to bi-allelic inactivation in tumors. To detect novel homozygous deletions in NSCLC, we began by applying our MCG Cancer Array-800 (5,6; http://www.cghtmd.jp/cghdatabase/index.html) for analysis of 27 NSCLC-derived cell lines.
Copy number gains and losses were seen to one degree or another in all NSCLC cell lines examined. The CGH array predicted frequent copy number gains (log2ratio >0.4 in >40% of cell lines) at 1q, 2p, 3q, 5p, 5q, 7p, 7q, 8q, 17q and 20q, and frequent losses (log2ratio <–0.4 in >40% of cell lines) at 8p, 9p, 10p, 13q, 18p, 18q and 19p. Table 1 lists the clones showing high-level amplifications (log2ratio >2) or homozygous deletions (log2ratio <–2). The results from CGH array analysis were mostly consistent with those of our earlier conventional CGH analysis of the same NSCLC cell lines (4,5), and were similar to published CGH results for primary NSCLC tumors (13).
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High-level amplifications were present in nine genes (clones), of which three were amplified in more than one cell line. We detected homozygous deletions in four genes (clones). Two genes located within 9p21.2, MTAP and CDKN2A/p16, were homozygously deleted in more than one cell line. Two others, PRDM2 (RIZ) at 1p36.21 and DBC1 (Fig. 1A) at 9q33.1, were both homozygously deleted in cell line 11-18. Although homozygous loss of DBC1 had never been documented in NSCLC before, it had been reported in urothelial cancers (7
–9), prompting us to examine whether DBC1 might be involved in the pathogenesis of lung tumors. The MCG Cancer Array-800 predicted not only complete loss of DBC1 (log2ratio=–3.4) in cell line 11-18, but also a hemizygous pattern of loss (log2ratio range: –0.4 to –2.0) in five other lines (18.5%, data not shown).
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Identification of target genes involved in homozygous deletion at 9q33.1
To identify all potential target genes for the homozygous deletion at 9q33.1 in cell line 11-18, we tried to define the region by fluorescence in situ hybridization (FISH), using 12 BACs as region-specific probes (Fig. 1B and C). No FISH signals specific for nine of those 12 BACs, including RP11-289D4, could be detected, whereas two others (BACs RP11-185G1 and RP11-57K1) showed only one signal each, and these signals were smaller than those produced in normal metaphase or interphase cells (data not shown). The results indicated that the proximal or distal breakpoints involved in the homozygous deletion lay on RP11-185G1 or RP11-57K1, respectively, on one allele at 9q33.1 in the 11-18 cell line (Fig. 1C). The estimated extent of the homozygous deletion was <1.5 Mb (closed green arrow in Fig. 1C); as information from the genome database (http://www.ncbi.nlm.nih.gov/ and http://genome.ucsc.edu/) indicated that DBC1 is the only gene within that region, it must represent the actual and sole target for homozygous loss in cell line 11-18. We then examined our NSCLC cell lines and laser-captured microdissection (LCM)-treated primary tumors for homozygous losses of DBC1 by means of genomic polymerase chain reaction (PCR), and again detected homozygous deletion of DBC1 in cell line 11-18 (1/27; 3.7%, Fig. 1D) as well as in two of 53 LCM-treated primary tumors (3.8%, Fig. 1E). Those results suggested that homozygous deletion represents a genetic mechanism for inactivating DBC1 in NSCLC cells, even though infrequent, and that this event was not an in vitro artifact that arose during the establishment of the cell lines.
Loss of DBC1 expression in NSCLC cell lines
Next we determined expression levels of DBC1 in NSCLC cell lines by reverse transcription (RT)–PCR. Of the 27 lines examined, not only the 11-18 cell line but also 21 lines without homozygous loss of DBC1 (21/26, 80.8%) lacked RT–PCR products (Fig. 1F). The other five NSCLC cell lines and normal lung did express DBC1 mRNA, suggesting that loss of expression in some tumors might result from mechanisms other than genomic deletion, including epigenetic events. Four of the five lines that had shown a hemizygous pattern of loss of DBC1 on our CGH array analysis failed to express the gene at all (data not shown).
Promoter activity of the DBC1 CpG island
Aberrant methylation of DNA in 5' regulatory regions harboring a higher than expected number of CpG dinucleotides is a key mechanism by which genes relevant to cancer initiation and progression can be silenced (14,15). A 1104 bp fragment around exon 1 of DBC1 (–609 to +494, Fig. 2A) was identified by means of the genome database (http://www.ebi.ac.uk/emboss/cpgplot/). Although hypermethylation in part of this CpG island (a 218 bp region containing 20 CpG sites, closed arrow almost covering region III in Fig. 2A; bisulfite map) was reported in urothelial cancers (7,9), no clear association had been established between the extent of methylation and the level of DBC1 mRNA (9). Therefore, we tested this CpG island for promoter activity, using several fragments encompassing or within the island (Fig. 2A) that were linked to the luciferase reporter in PC10 and SK-MES-1 cell lines lacking DBC1 expression. In both cell lines, a remarkable increase in transcriptional activity was a feature of all constructs containing regions I and II (fragments 1–3, Fig. 2A), whereas constructs without those fragments showed low transcriptional activity; therefore regions I and II, which do not contain the 218 bp region analyzed by Habuchi et al. (7,9), exert most of the promoter activity (Fig. 2B). When fragments within reporter constructs were methylated in vitro using SssI (CpG) methylase with S-adenosylmethionine (SAM), transcriptional activity was completely abolished (Fig. 2B). Similar results were obtained in other NSCLC cell lines, regardless of DBC1 expression (data not shown). Therefore, methylation of regions I and II in this CpG island appears to be important for repression of DBC1.
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Methylation of the DBC1 CpG island in NSCLC cell lines
To explore the potential role of methylation of this CpG island in silencing transcription of DBC1, we examined the methylation status of this gene in detail in NSCLC-derived cell lines by bisulfite-sequencing analysis using four PCR fragments covering 105 of the 108 CpG sites within the predicted CpG island. As shown in Fig. 2C, cells lacking DBC1 expression but without homozygous deletions of this gene were aberrantly hypermethylated, whereas hypomethylation was seen in DBC1-expressing cells. Hypermethylation was relatively frequent in regions I and II, in accord with the results of our promoter assay, although the degree of hypermethylation varied among CpG sites. The region analyzed in urothelial cancers was not hypermethylated in our NSCLC cell lines that did not express DBC1.
On the basis of the results of the bisulfite-sequencing analysis, we assessed the methylation status of the CpG island in all cell lines with or without expression of DBC1, by bisulfite-PCR experiments using appropriate restriction enzymes for each of the four regions (Fig. 2D). Nineteen of the 21 cell lines lacking DBC1 expression without homozygous loss showed hypermethylation (>20%), (5) in any of eight region–enzyme combinations, whereas all five lines with DBC1 expression showed low methylation (
20%) in all of the region–enzyme combinations (Fig. 2D and data not shown). These results were consistent with those of the original bisulfite-sequencing analysis: CpG sites in regions I and II of the CpG island tended to be extensively methylated, and their methylation status was likely to be inversely correlated with expression of DBC1. Taken together, the findings suggested that methylation of part of the DBC1 CpG island was closely related to transcriptional silencing of DBC1 in NSCLC cells without homozygous loss of this gene.
Effect of demethylation by 5-aza 2'-deoxycytidine on DBC1 expression
To investigate whether demethylation could restore expression of DBC1 mRNA, we treated NSCLC cells with 5-aza-dCyd, a methyltransferase inhibitor, for 5 days. Induction of DBC1 mRNA occurred after treatment with 5 or 10 mM of 5-aza-dCyd in cells lacking DBC1 expression (NCI-H460 and LU99A, Fig. 2E).
A growing body of data indicates that histone modification, including hypoacetylation of histones (16), is involved in the gene silencing caused by methylation of DNA (17,18). To evaluate the role of histone acetylation in our NSCLC lines, we tested whether interference with the activity of histone deacetylases alone or in combination with DNA methyltransferases might lead to reactivation of the silent genes. Cells were treated with trichostatin A (TSA), a histone deacetylase inhibitor, and/or 5-aza-dCyd. Treatment with TSA alone had no affect on expression of DBC1 in LU99A cells, whereas we detected an elevation in DBC1 expression in NCI-H460 cells after treatment with TSA alone. In both cell lines, TSA synergistically enhanced the effect of 5-aza-dCyd on expression of DBC1 (Fig. 2E). Those results indicate some role for histone deacetylation in silencing of DBC1 among methylated NSCLC cells.
Methylation of the DBC1 CpG island in primary NSCLC tumors
To determine whether aberrant methylation of DBC1 also takes place in primary NSCLCs, we carried out methylation analysis in a panel of 65 primary tumors; corresponding normal lung tissues were available for 14 of them (Table 2). As shown in Figure 3A and B, the DBC1 CpG island was hypermethylated in primary NSCLCs when compared with normal lung tissues, with a pattern of methylation similar to that seen in NSCLC cell lines. As those DNAs had been isolated from snap-frozen tumors rather than LCM-treated samples because bisulfite treatment requires larger amounts of high-quality DNA, the lower methylation density in primary tumors when compared with cell lines could reflect unavoidable contamination of the specimens with noncancerous cells, leading to underestimation. Notably, even though RNAs for expression analysis were available in only three of 14 paired cases, two hypermethylated tumors (case 385 and 420, Fig. 3A and B) showed much lower expression of DBC1 than their normal-tissue counterparts (Fig. 3C), suggesting that selective hypermethylation of the DBC1 promoter region often contributes to the silencing of this gene in NSCLCs.
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Association between clinicopathological characteristics and methylation of the DBC1 CpG island in primary cases
To clarify the importance of methylation of the DBC1 CpG island in the pathogenesis of NSCLC, we first analyzed the relationship between methylation status and clinicopathological characteristics of all 65 primary tumors (Table 3). Total percentage of intensities of methylated alleles in eight fragment–enzyme combinations was defined as the methylation status of the DBC1 CpG island in each sample, which was not associated with histologic subtype or tumor staging. On the other hand, methylation of the DBC1 CpG island occurred more frequently in men, elderly patients and smokers than in women, younger patients and nonsmokers respectively. Although the pattern of methylation within eight fragment–enzyme combinations in tumors was similar between smokers and nonsmokers, methylation in regions I–III was more frequent in tumors from smokers than from nonsmokers (Fig. 3D).
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Since CpG islands upstream of some tumor suppressor genes can become hypermethylated in normal epithelial cells in response to factors such as exposure to tobacco and aging, we used bisulfite-PCR to analyze methylation of the 14 tumors for which paired normal lung tissues were available. The CpG island of DBC1 was methylated in paired normal lung tissues, but to a much lesser degree than in the tumors (Table 3, Fig. 3E). The pattern of methylation within the eight fragment–enzyme combinations in normal lung tissues was similar to that in paired tumors, but normal tissues showed no methylated alleles in fragments III–HhaI or III–BstUI (Fig. 3A and E), suggesting that methylation of those particular sites might be tumor-specific. In normal lung tissues the levels of methylation within all eight fragment–enzyme combinations were higher in older patients and smokers than in younger patients and nonsmokers, respectively, although the differences did not reach statistical significance (Table 3). However, the number of paired samples was too limited to allow any firm conclusions on that point.
Suppression of cell growth after restoration of DBC1 expression
To investigate whether restoration of DBC1 expression would suppress growth of NSCLC cells in which the gene had been silenced, we performed colony formation assays (6,19) using the full coding sequence of DBC1. As shown in Figure 4,2 weeks after transfection and subsequent selection of drug-resistant colonies, the numbers of large colonies produced by DBC1-transfected cells decreased compared with cells containing empty vector. However, as predicted from experiments by Wright et al. (20), we have never been able to obtain stably expressed, detectable levels of epitope-tagged DBC1 protein by constitutive or inducible strategies (data not shown). Our preliminary study also failed to construct an adenovirus-mediated expression system (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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We performed CGH array analysis to map genome-wide DNA copy number alterations in a panel of 27 NSCLC cell lines. Since the most common chromosomal imbalances had previously been identified by conventional CGH in these cell lines (21
), we focused on the more extreme form of chromosomal imbalance, homozygous deletion, which is impossible to detect by conventional means. Using our custom-made MCG Cancer Array-800 (5,6), we identified a homozygous deletion of DBC1 (9q33.1) in one NSCLC cell line. Deletion of that gene had been reported in urothelial cancer (7), but never before in NSCLC. Subsequently, precise mapping clearly showed that DBC1 was the only gene located in the region homozygously deleted in that cell line. Since relatively frequent losses of heterozygosity at 9q had been reported in NSCLCs (10–12) but no putative targets involved in the pathogenesis of NSCLC had been identified there, we investigated whether DBC1 might function as a tumor suppressor in human lung tissue.
We also observed homozygous deletions of DBC1 in two of 53 primary NSCLCs (adenocarcinomas). As no mutation of this gene had been identified in urothelial tumors (7), genomic alterations leading to inactivation of DBC1 appear to be rare events. On the other hand, we observed silencing of DBC1 mRNA expression in 21 of 26 other NSCLC cell lines without homozygous deletion of this gene (80.8%), suggesting that DBC1 might be silenced mainly in other ways in NSCLC cells, such as by epigenetic mechanisms. Although many genes involved in carcinogenesis are inactivated by aberrant methylation of CpG islands in their promoter regions, the methylation status of the DBC1 CpG island had been investigated in only a few types of tumor (7,22), and any correlation between expression of DBC1 and the methylation status of its CpG island remained unclear (9).
Moreover, in those reported studies the region analyzed was only part of the DBC1 CpG island; the critical region for promoter activity was never determined. Therefore, we performed fine mapping of the region showing promoter activity, investigated the frequency of methylated CpG sites throughout almost the entire CpG island of DBC1 and compared the methylation status of various fragments with expression of DBC1 mRNA. Hypermethylation of CpG sites occurred in a region upstream of exon 1 that exhibited promoter activity, and the hypermethylation seemed to be inversely correlated with expression of DBC1 mRNA in NSCLC cell lines. The same observations pertained among primary NSCLC tumors for which paired normal lung tissues were available for comparison. Those results suggested that the region previously analyzed in other types of tumor (region III) (7–10,21) was likely to be outside the frequently methylated region that is highly associated with DBC1 silencing, even though DNA methylation in region III was observed in a tumor-specific manner and further examination will be needed to determine the clinical significance of this region in terms of diagnosis of NSCLC. Since our CGH array analysis of NSCLC cell lines revealed that some lines without DBC1 homozygous deletion exhibited hemizygous losses of this gene, most ‘two hit’ DBC1-inactivating events in NSCLC are likely to be either (a) deletion of one allele and methylation of the other or (b) methylation of both alleles. In any case, those losses might well be involved in the pathogenesis of this disease.
Among our panel of 65 primary NSCLC tumors, hypermethylation of the DBC1 CpG island was correlated with gender, age and habitual smoking, but not with tumor stage or histology. At present, the molecular mechanisms responsible for differential susceptibility to hypermethylation during aging remain unclear, but they are probably related to several factors, namely de novo DNA methyltransferase activity, modulating factors such as smoking, different degrees of protection against methylation and local triggering factors such as an unusual secondary structure around a methylation center. Although we have no direct evidence that smoking induces DNA methylation, recent reports have suggested that smoking increases DNA methyltransferase activity and that DNA hypermethylation is associated with exposure to tobacco smoke (23,24). Therefore, the increased prevalence of hypermethylation of the DBC1 promoter in normal lung tissue of smokers as well as in their tumors may result from increased methyltransferase activity.
In our analysis of paired samples, noncancerous lung tissues showed a similar pattern of methylation as in tumors but with a lesser degree of methylation. Those findings were consistent with results reported for urothelial cancer (9) and oral squamous cell carcinoma (22), suggesting that methylation of the DBC1 CpG island may represent an early event in malignant development of NSCLC and other tumors. Gene-specific, age- and smoking-related methylation in normal bronchial epithelium has been reported recently (25). Longitudinal prospective studies and quantitative assays will help to clarify the role of DBC1 gene silencing during development and progression of NSCLC, and determine the clinical significance this alteration in terms of screening high-risk groups of people (26).
Colony formation assays using DBC1-transfected NSCLC cell lines demonstrated growth-suppressing and/or antiproliferative activity of the DBC1 protein, confirming findings of Nishiyama et al. (27) in bladder-cancer cell lines. However, little information is available regarding the functional consequences of DBC1 loss in terms of carcinogenesis. Cell-cycle studies have suggested that DBC1 modulates the G1 checkpoint (27); in those experiments, overexpression of DBC1 caused a slower G1 transition rather than G1 arrest and did not affect apoptosis. On the other hand, Wright et al. (20) demonstrated that DBC1 induces cell death, not of the classic apoptotic type, in cultured tumor cells. As no system we tried was successful for obtaining stable cell lines expressing DBC1, including Tet-on/off inducible-lines, it will be difficult to investigate the mechanisms of tumor-suppressing activity of DBC1 any further. However, its tumor-suppressing activity in vitro, together with (a) DBC1 expression in normal lung and (b) correlation between a high rate of hypermethylation in the DBC1 promoter and expression of DBC1 mRNA, support the candidacy of this gene as a tumor suppressor for NSCLC. Additional studies will be required to unravel the carcinogenic consequences of loss of DBC1 function in human lung tissue.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Cell lines and primary tumors
Of the 27 NSCLC cell lines employed, 11 were derived from squamous cell carcinomas (EBC1, HS-24, LK-2, PC10, HUT15, VMRC-LCP, Lc-1 sq, ACC-LC-73, SK-MES-1, Sq-1 and KNS-62), 10 from adenocarcinomas (11-18, A549, ABC-1, RERF-LC-MS, RERF-LC-OK, VMRC-LCD, PC14, HUT29, SK-LC-3 and RERF-LC-KJ) and six from large cell carcinomas (86-2, LU65, PC13, ACC-LC-33, NCI-H460 and LU99A). Cells were maintained in appropriate media (RPMI-1640 or Dulbecco's modified Eagle's medium) supplemented with 10% fetal bovine serum and 100 units/ml penicillin/100 µg/ml streptomycin.
Primary tumor samples were obtained during surgery from 118 patients being treated at the National Cancer Institute Hospital in Tokyo or the Hokushin General Hospital in Nagano, Japan, with written consent from each patient in the formal style and after approval by the local ethics committees. Samples from 53 patients with adenocarcinoma were paraffin-embedded for LCM after fixation in methanol for 24 h, as described elsewhere (28); tumors from the other 65 patients (Table 2), along with adjacent noncancerous lung tissues from 14 of them, were frozen immediately in liquid nitrogen and stored at –80°C until required. None of the patients had received preoperative radiation, chemotherapy or immunotherapy. No survival analysis was performed because the follow-up period was too short and the number of deaths or recurrences after surgery was too small for prognosis to be evaluated.
CGH array analysis
Our MCG Cancer Array-800 (5,6) contains 800 BAC/PAC clones carrying genes or sequence-tagged site markers of potential importance in cancer genesis or progression (http://www.cghtmd.jp/cghdatabase/index.html). Hybridizations were carried out as described elsewhere (6). Briefly, test and reference DNAs were labeled, respectively, with Cy3- or Cy5-dCTP (Amersham Biosciences, Tokyo, Japan), precipitated together with ethanol in the presence of Cot-1 DNA, re-dissolved in a hybridization mix [50% formamide, 10% dextran sulfate, 2x SSC, 4% sodium dodecyl sulfate (SDS), pH 7], and denatured at 75°C for 10 min. After pre-incubation at 37°C for 30 min, each mixture was applied to array slides and incubated at 42°C for 48–72 h. After hybridization, the slides were washed once in a solution of 50% formamide, 2x SSC (pH 7.0) for 15 min at 50°C, once in 2x SSC, 0.1% SDS for 15 min at 50°C, and once in a 0.1 mol/l sodium phosphate buffer containing 0.1% Nonidet P-40 (pH 8) for 15 min at room temperature, then scanned with a GenePix 4000B (Axon Instruments, Foster City, CA, USA). Acquired images were analyzed with GenePix Pro 4.1 imaging software (Axon Instruments). Fluorescence ratios were normalized so that the mean of the middle third of log2ratios across the array was zero. Average ratios that deviated significantly (>2 SD) from zero were considered abnormal.
FISH
Metaphase chromosomes were prepared from normal male lymphocytes and from each NSCLC cell line. FISH analyses were performed as described previously (29), using BACs located around the region of interest as probes.
Screening for homozygous deletions by genomic PCR using cell lines and LCM samples
Methanol-fixed, paraffin-embedded tissues were prepared for LCM with a PixCell II LCM system (Arcturus Engineering, Mountain View, CA, USA). Genomic DNA was isolated in lysis buffer (10 mM Tris–Hcl at pH 7.5, 1 mM ethylene diamine tetraacetic acid, 0.5% SDS) and amplified by adaptor-ligation-mediated PCR after end-filling, as described by Tanabe et al. (30).
We screened DNAs from 27 cell lines and 53 primary NSCLCs (adenocarcinomas) for homozygous losses by genomic PCR. All primer sequences are available on request.
RT–PCR
Single-stranded cDNAs were generated from total RNAs using the SuperScriptTM First-Strand Synthesis System (Invitrogen, Carlsbad, CA, USA), and amplified with primers specific for the DBC1 gene. The glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) was amplified at the same time to allow estimation of the efficiency of cDNA synthesis.
Promoter constructs, in vitro methylation and promoter reporter assay
A 1104 bp fragment (region 1 in Fig. 2A) of a CpG island in DBC1 predicted by the CpGPLOT program (http://www.ebi.ac.uk/emboss/cpgplot/) and three other fragments (Fig. 2A, regions 2–4) around DBC1 exon 1 were obtained by PCR. To examine the effect of methylation on CpG sites, these fragments were treated overnight with SssI (CpG) methylase (3 units/µg of DNA) in the presence (methylated) or absence (unmethylated) of 1 mM SAM. Complete methylation of the DNA fragment was confirmed by digestion with TaqI or HhaI. Each fragment was ligated into the pGL3-Basic vector (Promega, Madison, WI, USA), purified on gels, and used directly for transfection. Equal amounts of constructs containing either methylated or unmethylated fragments were introduced into cells with an internal control vector (pRL-hTK, Promega), using FuGENE6 (Roche Diagnostics, Tokyo, Japan). A pGL3-Basic vector without insert served as a negative control. Firefly luciferase and Renilla luciferase activities were each measured 36 h after transfection by means of the Dual-Luciferase Reporter Assay System (Promega); relative luciferase activities of the samples were calculated and normalized versus Renilla luciferase activity.
Bisulfite-PCR analysis and bisulfite sequencing
Genomic DNAs from frozen samples were treated with sodium bisulfite using an EZ DNA Methylation kit (Zymo Research, Orange, CA, USA), and subjected to PCR using primer sets designed to amplify the CpG island of interest.
For bisulfite-PCR analysis, PCR products were digested with TaqI, HhaI, MluI or BstUI, which recognize sequences unique to the methylated (bisulfite-unconverted) alleles but cannot recognize unmethylated (bisulfite-converted) alleles, and electrophoresed. After the gels were stained with ethidium bromide, the %intensities of methylated alleles were calculated by densitometry using MultiGauge 2.0 (Fuji film, Tokyo, Japan). For bisulfite sequencing, PCR products were subcloned and then sequenced.
Drug treatment
Cells were treated with varying concentrations of 5-aza-dCyd for 5 days, and/or 100 ng/ml TSA for varying periods. For the synergistic study, 5 mM of 5-aza-dCyd was present in the cultures for 5 days, and/or 100 ng/ml TSA was added for the last 12 h.
Transient transfection, western blotting and colony formation assays
A plasmid expressing Myc-tagged DBC1 (pCMV-Tag3-DBC1), was obtained by cloning the RT–PCR product of the full coding sequence of DBC1 into the pCMV-Tag3 eukaryotic expression vector (Stratagene, La Jolla, CA, USA) in-frame along with the Myc-epitope. pCMV-Tag3-DBC1, or the empty vector (pCMV-Tag3-mock) control, were transfected into cells for colony formation assays as described elsewhere (6,19). Expression of DBC1 protein in transiently transfected cells was confirmed 48 h after transfection, by western blot analysis using anti-Myc antibody (Cell Signaling Technology, Beverly, MA, USA) as described elsewhere (29). After 2 weeks of incubation in six-well plates with appropriate concentrations of G418, cells were fixed with 70% ethanol and stained with crystal violet.
Statistical analysis
The Wilcoxon matched-pairs signed-ranks test was used to compare the %intensities of methylated alleles between 14 paired normal and tumor samples. Group mean comparisons of the %intensities of methylated alleles were performed with Mann–Whitney U test or Kruskal–Wallis test. P<0.05 was required for significance in each case.
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ACKNOWLEDGEMENTS |
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We are grateful to Professor Yusuke Nakamura (Human Genome Center, Institute of Medical Science, The University of Tokyo) for continuous encouragement throughout this work. We also thank Professor Takashi Takahashi (Laboratory of Cancer Cell Biology, Research Institute for Disease Mechanism and Control, Nagoya University Graduate School of Medicine), Professor Akira Horii (Department of Molecular Pathology, Tohoku University Graduate School of Medicine) and Professor Takehiko Fujisawa (Department of Thoracic Surgery, Chiba University Graduate School of Medicine) for providing NSCLC cell lines, and Ai Watanabe for technical assistance. This work was supported by grants-in-aid for Scientific Research (to J.I., I.I., S.Y.) and Scientific Research on Priority Areas (C) (to J.I., I.I.) and a Center of Excellence Program for Research on Molecular Destruction and Reconstruction of Tooth and Bone (to J.I.) from the Ministry of Education, Culture, Sports, Science, and Technology, Japan; and a grant from Core Research for Evolutional Science and Technology (CREST) of Japan Science and Technology Corporation (JST) (to J.I.).
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REFERENCES |
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