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Am J Pathol. Jul 2003; 163(1): 37–45.
PMCID: PMC1868173

Methylation Target Array for Rapid Analysis of CpG Island Hypermethylation in Multiple Tissue Genomes

Abstract

Hypermethylation of multiple CpG islands is a common event in cancer. To assess the prognostic values of this epigenetic alteration, we developed Methylation Target Array (MTA), derived from the concept of tissue microarray, for simultaneous analysis of DNA hypermethylation in hundreds of tissue genomes. In MTA, linker-ligated CpG island fragments were digested with methylation-sensitive endonucleases and amplified with flanking primers. A panel of 468 MTA amplicons, which represented the whole repertoire of methylated CpG islands in 93 breast tumors, 20 normal breast tissues, and 4 breast cancer cell lines, were arrayed on nylon membrane for probe hybridization. Positive hybridization signals detected in tumor amplicons, but not in normal amplicons, were indicative of aberrant hypermethylation in tumor samples. This is attributed to aberrant sites that were protected from methylation-sensitive restriction and were amplified by PCR in tumor samples, while the same sites were restricted and could not be amplified in normal samples. Hypermethylation frequencies of the 10 genes tested in breast tumors and cancer cell lines were 60% for GPC3, 58% for RASSF1A, 32% for 3OST3B, 30% for HOXA5, 28% for uPA, 25% for WT1, 23% for BRCA1, 9% for DAPK1, and 0% for KL. Furthermore, hypermethylation of 5 to 7 loci of these genes was significantly correlated with hormone receptor status, clinical stages, and ages at diagnosis of the patients analyzed. This novel approach thus provides an additional avenue for assessing clinicopathological consequences of DNA hypermethylation in breast cancer.

CpG islands are short stretches of guanine cytosine (GC)-rich sequences frequently located in the promoter and the first exon of genes. 1 In cancer cells, aberrant DNA methylation is known to occur within these regulatory regions. This epigenetic event is somatically heritable and increased density of methylated CpG sites within a promoter region can accumulate over time during tumorigenesis. 2,3 The methylation build-up is associated with the formation of repressive chromatin and the resulting silencing of the associated gene. 3 Accumulated evidence indicates that this epigenetic mechanism is responsible for silencing >100 genes for tumorsuppression, genomic instability and repair, apoptosis, cell cycle regulation, and signal transduction in many tumor types. 4 Hypermethylated CpG islands therefore play a causal role in promoting tumor development and are useful molecular markers for cancer diagnosis and prognosis.

To dissect complex epigenetic patterns in cancer cells, we previously developed a microarray-based approach in which thousands of short CpG island tags known as probes were arrayed on nylon membranes or glass slides. 5-7 DNA targets representing different pools of methylated CpG islands in tumor or control samples were used to hybridize the microarray panel. Hypermethylated CpG islands were identified in tumor samples based on their differential hybridization patterns relative to normal controls. This genome-wide screening tool provides an efficient way to survey DNA hypermethylation in one tumor at a time and has been used to identify many novel methylation-silenced genes. 5-7 To further assess the utility of candidate CpG islands as epigenetic markers for tumor progression, we reversed the process in this study by arraying multiple methylation targets on solid supports and hybridizing the array one at a time with different CpG island probes. Similar to the concept of tissue microarray, 8 this approach, called Methylation Target Array (MTA), has the capacity for simultaneous interrogation of CpG island hypermethylation in hundreds of clinical samples. Here we describe its application in determining hypermethylation profiles of 10 genes in multiple breast tumors. These profiles were correlated with the clinicopathological features of the patients.

Materials and Methods

Tissue Samples and Cell Lines

Tumor tissues were obtained from patients undergoing mastectomy before chemotherapy at the Ellis Fischel Cancer Center in compliance with the Institutional Review Board. Clinicopathological features were determined based on the Tumor-Node-Metastasis system. 9 All breast tumors analyzed in this study were infiltrating ductal carcinomas. Adjacent normal parenchyma 3 to 5 cm away from the tumor area was obtained to serve as a normal breast tissue control. The estrogen receptor (ER)/progesterone receptor (PR) status of clinical samples was determined by either the dextran-coated charcoal assay or the immunoperoxidase technique, as described. 10 Based on our estimate, 5% to 10% of stroma or infiltrating lymphocytes are present in the collected tumor specimens. These were considered as part of the “texture” of breast tumors, which were used throughout this study without further microdissection to procure pure tumor cells. The presence of residual normal cells was not expected to affect the overall interpretation of the methylation data subsequently acquired. Tumor specimens contaminated with significant amounts of normal cells were not used in the study. Breast cancer cell lines T47-D, ZR-75, Hs578t, and MDA-MB-231 were maintained as described. 5 Genomic DNA was isolated using the QIAamp Tissue Kit (Qiagen, Valencia, CA).

Construction of Methylation Target Array

To generate methylation targets, genomic DNA (1 μg per sample) of multiple tissue samples was individually placed in 96-well format microtubes and digested with MseI, BfaI, NlaIII, or Tsp509I (25 U/well; New England Biolabs, Beverly, MA). The digests were then purified and their sticky ends ligated with 0.5 nmol of unphosphorylated primer linkers (Table 1) [triangle] . The ligated DNA was purified and further digested with methylation-sensitive endonucleases, BstUI (25 U/well, 60°C, 16 hours) and HpaII (25 U/well, 37°C, 16 hours), successively. PCR (300 μl per tube) was performed in the 96-well format microtubes using the ligated DNAs as templates and a linker as primer, and subjected to 20 cycles of amplification. Amplified targets were concentrated and printed (~0.3 μl per dot, 0.5 μg/μl) as duplicate microdots onto nylon membranes using the MULTI-PRINT replicator (V&P Scientific, San Diego, CA). Spotted DNA was denatured and UV-crosslinked to the matrix.

Table 1.
Primers for Linker-PCR, MTA Probes, Methylation-Specific PCR, and Reverse-Transcription PCR

MTA Hybridization

Primer sequences and PCR conditions used to prepare probes are listed in Table 1 [triangle] . PCR-generated probes were purified, labeled with 32P-dCTP, and hybridized with MTA filters at high stringent conditions (68 to 72°C) using the Rapid-Hyb Buffer system (Amersham Biosciences, Inc., Piscataway, NJ). Post-hybridization washing was performed as previously described. 5 The hybridized filters were exposed in a PhosphorImager (Amersham Biosciences) for 1 to 7 days. Each MTA experiment was repeated 1 or 2 times. Positive hybridization signals, which are indicative of CpG island methylation in targets, were first visually scored. Because signal intensities of hybridized targets varied due to different amounts of DNA dotted on the membrane, individual spots were quantified using ImaGene 4.0 (BioDiscovery, Los Angeles, CA). The signal intensity of each spot was then normalized with that of an internal control (ie, the Cot-1 hybridized spot). A positive signal for CpG island methylation was again scored when a cutoff intensity [(signal − background) × normalized factor] was greater than 30 (an arbitrary unit) in duplicate spots of an arrayed target. Using this cutoff, the level of concordance between the two scoring methods was ≥95%.

Methylation-Specific PCR

Genomic DNA (1 to 2 μg) was treated with sodium-bisulfite according to the manufacturer’s recommendations (CpGenome; Intergen, Purchase, NY) and amplified with specific p16INK4 primers (Table 1) [triangle] for methylated or unmethylated DNA. All PCR reactions were performed on PTC-100 thermocyclers (MJ Research, Watertown, MA) and in 25-μl volumes using the AmpliTaq system (Applied Biosystems, Foster City, CA). PCR products were separated on 1.0% agarose gels.

Demethylation Treatment and Northern and Reverse Transcription (RT)-PCR Assays

Breast cancer cell lines were cultured in the absence or presence of 5-aza-2′-deoxycytidine (DeoxyC, 750 nmol/L) for 6 days and harvested for RNA isolation using the RNAeasy system (Qiagen). Ten μg of RNA from treated and untreated samples were electrophoresed on a 1.5% agarose gel and subjected to northern analysis using a 3OST3B cDNA probe as described. 5 RT-PCR was conducted using primers (Table 1) [triangle] located at the 3′-ends of 3OST3B. The levels of 3OST3B mRNA were normalized with the level of β-actin mRNA.

Statistical Analysis

A comparison of methylation frequencies across clinicopathological parameters was performed using the X 2 test or Fisher’s exact method. The Mann-Whitney U nonparametric test was used to compare the coordination of DNA methylation in multiple CpG island loci. A P value less than 0.05 was defined as being statistically significant.

Results and Discussion

Rationale and Strategy of MTA

An initial study was conducted to determine whether the MTA strategy would prove feasible to assess CpG island hypermethylation in multiple breast tumors. To prepare targets, we restricted DNA samples with 4-base frequent cutters that cut elsewhere in the genome (<0.2-kb), but preserve as much as possible the integrity of CpG islands. Four such endonucleases, MseI (T↓TAA), Tsp509I (↓AATT), NlaIII (CATG↓), and BfaI (C↓TAG), whose recognition sequences were shown to be infrequent within GC-rich fragments, 11 are ideal for use in the digestion of the genome. We independently surveyed 100 genes frequently hypermethylated in various cancers and showed that DNA fragments (0.2- to 2.0-kb) restricted by these endonucleases are expected to encompass GC-rich sequences of these genes (Table 2) [triangle] . After digestion, the flanking cut sites of GC-rich fragments were ligated to linkers and then sequentially restricted with 2 methylation-sensitive endonucleases, BstUI and HpaII, the recognition sites of which are always present in CpG islands (see examples in Table 2 [triangle] ). The digest was used as template for PCR amplification with a linker-primer. DNA fragments containing methylated recognition sites were protected from the restriction and were amplified by PCR, while unmethylated or partially methylated fragments were cut away and could not be amplified. Therefore, the MTA technique is limited to the rapid screening of genes having completely methylated restriction sites in the region of interest. Samples with loci having partial methylation in these sites will not be detected by this technique.

Table 2.
Survey of Internal Methylation-Sensitive Sites in 100 CpG Islands Frequently Hypermethylated in Various Cancers

Incomplete methylation-sensitive digestion might occur during sample preparation, resulting in false-positive detection of DNA methylation after PCR amplification. We therefore tested the efficiency of digestion in two CpG island fragments (BRCA1 and p16INK4) known to be unmethylated in normal blood DNA. Primers flanking an NlaIII fragment of BRCA1, which contains 2 internal HpaII and 3 BstUI sites, were used for PCR amplification (see Figure 3 [triangle] ). Primers were also designed for amplification of an NlaIII fragment in p16INK4. We then determined at which cycles of PCR the amplification of undigested DNAs became apparent. As shown in Figure 1A [triangle] , at ≥29 cycles of PCR the amplified BRCA1 or p16INK4 fragment was seen in the digested samples in ethidium bromide-stained gels, suggesting that the false finding of DNA methylation could occur when high cycles of amplification are used in sample preparation. We therefore used fewer PCR cycles (20 cycles) in the MTA assay to prevent unwanted amplification of residual undigested DNA. This strategy was further tested by amplifying DNA fragments with specific primers derived from 9 CpG islands known to be hypermethylated in cancer (Figure 1B) [triangle] . As shown, methylated fragments were preferentially amplified in the pooled targets of 20 to 30 breast tumors, but not in those of normal controls. No amplification was detected in a control CpG island, KL, in either tumor or control.

Figure 1.
A: Methylation-sensitive restriction test. The efficiency of digestion in CpG fragments in BRCA1 and p16INK4 was examined by subjecting duplicate tubes of genomic DNA, one doubly-digested with BstUI and HpaII (D) and the other mock-digested without enzymes ...
Figure 3.
Map locations of restriction sites in the 10 genes analyzed by MTA. The shaded horizontal bar indicates that the first exon region and its position designation is relative to the transcription start site (+1). The short vertical bars indicate ...

We next determined whether repetitive sequences might be overpopulated in the amplified target pools, leading to decreased sensitivity in the detection of uniquely methylated CpG islands in MTA. To determine the content of repetitive sequences after amplification, serial dilutions of 4 target pools (MseI, Tsp509I, NlaIII, and BfaI) and the human repetitive Cot-1 DNA (used as a control) were equally dotted onto nylon membrane and hybridized with a Cot-1 probe (Figure 2A) [triangle] . Between 22% and 34% of repetitive sequences were calculated to be included in the methylation targets (Figure 2B) [triangle] . This amount is lower than that of the unprocessed genome (40% to 50%) and therefore would not be expected to interfere in the MTA hybridization with specific probes.

Figure 2.
The content of repetitive sequences in different methylation targets. A: Serial dilution of different cutter-generated (MseI, Tsp509I, NlaIII, or BfaI) methylation targets (15 to 500 ng) and an identical amount of human Cot-1 DNA were dotted on the nylon ...

MTA Analysis of CpG Island Hypermethylation in Breast Cancer

MTA was initially used to survey hypermethylation of the p16INK4 CpG island in breast cancer. Methylation targets (NlaIII fragments) prepared from 93 breast tumors, 4 breast cancer cell lines, and 20 normal breast tissues were arrayed onto nylon membrane and hybridized with a 219-bp p16INK4 probe (Figure 3 [triangle] and Figure 4A [triangle] ). The nylon membrane was also hybridized with a Cot-1 control probe, which was used to determine the relative amounts of DNA dotted on the array. Positive hybridization signals, indicative of DNA methylation, were first visually scored on the MTA membrane and later verified with the calculated signal intensity of each spot after normalization with that of the Cot-1 control (see the scoring method described in Materials and Methods). The results showed that 22.6% (22 of 97) of breast tumors and cell lines were positive for methylation. None of the 20 normal samples, however, showed detectable methylation. To validate these methylation findings, we performed methylation-specific PCR (MSP) on 36 tumor samples (see representative examples in Figure 4B [triangle] ). Except for one tumor (T23), the MSP assay of the rest of the samples confirmed the MTA results. The discrepancy of T23 can be attributed to different methodologies used in the methylation analysis. Alternatively, it suggests a low level of false-positive findings by MTA, which may not interfere with methylation screening in a large number of tissue samples.

Figure 4.
A: Methylation Target Array (MTA). The MTA chart is used to indicate the location of each arrayed target. T, tumor; N, normal control; Pos, positive control, ie, target prepared without methylation-sensitive restriction; and Neg, negative control, ie, ...

We therefore expanded the MTA analysis to an additional 9 genes, RASSF1A, uPA, HOXA5, DAPK1, WT1, KL, BRCA1, GPC3, and 3OST3B. Their CpG islands are found to be located in the promoter and the first exon of the genes. Except for 3OST3B, the 4-base cutter fragments to be analyzed in MTA are located near or within the promoter and first exon of these genes (Figure 3) [triangle] . The NlaIII fragment of 3OST3B is located ~2.5-kb upstream of its first exon, a region previously cloned by the ICEAMP technique developed by us 12 for the identification of aberrantly-methylated DNAs in cancer. Representative results of MTA hybridization are shown in Figure 4C [triangle] . CpG island hypermethylation was scored and the results were summarized in Figure 5A [triangle] . The overall methylation frequencies detected in breast tumor and cell lines were 60% for GPC3, 58% for RASSF1A, 32% for 3OST3B, 30% for HOXA5, 28% for uPA, 25% for WT1, 23% for BRCA1, and 9% for DAPK1. Methylation of the KL CpG island in these tumors was not detected by MTA. Eighty-seven percent (81 of 93) of these tumors had hypermethylation in at least one of the 10 genes studied. Furthermore, hypermethylation of multiple genes (5 to 7 loci) was observed in 20% of the tumors. The 4 breast cancer cell lines had an overall higher frequency of hypermethylation in these genes than that of primary tumors. While only one normal sample showed methylated BRCA1 promoter, the rest of the 19 normal controls had no detectable hypermethylation by MTA.

Figure 5.
Profiles of Methylation Target Array in breast cancer. A: The methylation status of 10 promoter CpG islands in 93 breast tumors, 4 breast cancer cell lines, and 20 normal breast tissues. A filled box indicates the presence of methylation and an open box ...

These methylation findings are reminiscent of those previously reported in breast cancer. The high frequencies (>50%) of CpG island hypermethylation detected in GPC3 and RASSF1A genes are consistent findings in breast cancer. 7,13,14 On the other hand, hypermethylation of the DAPK1 gene is less frequent (<20%) in breast tumors, at a rate similar to those reported. 4,15 The frequency (23%) of BRCA1 hypermethylation reported here is somewhat higher than that (13%) of a previous survey. 4 The median rates of hypermethylation in WT1 and p16INK4 genes are also close to the reported incidence in breast cancer. 4,16 While CpG island hypermethylation in HOXA5 was previously reported in 80% of p53-negative breast tumors, 17 the overall rate of methylation shown here is 30%. The methylation frequency of uPA has previously not been reported in primary tumors, but hypermethylation of this gene was shown to result in transcriptional silencing in breast cancer cell lines. 18

Hypermethylation of the upstream region of 3OST3B is a novel finding in breast cancer. This gene encodes 3-O-sulfotransferases known to modulate the sulfonation of heparan side chains needed in a wide range of biological processes. 19 We further correlated this aberrant methylation with the transcriptional activity of 3OST3B in breast cancer cell lines (Figure 6,A and B) [triangle] . Hypermethylation of the 3OST3B promoter was detected in MDA-MB-231 and expression analyses revealed that this hypermethylation was associated with down-regulation of 3OST3B, and was reactivated on the treatment of a demethylation agent, DeoxyC. The expression of 3OST3B was also restored in breast cancer cell line T47-D (Figure 6C) [triangle] . No methylation of 3OST3B was detected in ZR-75 and Hs578t (data not shown) and their expression levels of 3OST3B were not influenced by the demethylation treatment. Our future studies will focus on methylation analysis of this gene promoter and the functional consequences of its methylation-mediated silencing in relation to breast tumorigenesis.

Figure 6.
Methylation and expression analysis of the 3OST3B gene. Breast cancer cell lines were cultured in the absence or presence of 5-aza-2′-deoxycytidine (DeoxyC; 750 nmol/L) for 6 days and harvested for DNA and RNA isolation. A: Methylation-specific ...

Association of Hypermethylation Profiles with Clinicopathological Features of Patients

The frequencies of CpG hypermethylation in the primary breast tumors analyzed were found to be associated with several clinicopathological features of the patients (Table 3) [triangle] . Hypermethylation of multiple loci (ie, 5 to 7 loci) was significantly correlated with ER/PR-negative (P = 0.002) or late-stage (P = 0.02) tumors (Figure 7,A and B) [triangle] . Hypermethylation in the 9 CpG islands assayed by MTA also occurred less frequently in older patients (≥70 years old) with breast cancer (P = 0.025) (Figure 7C) [triangle] .

Figure 7.
Correlation of CpG island hypermethylation with clinicopathological features of breast tumors. The x axis indicates the total number of hypermethylated loci (low for 0 to 2 loci, median for 3 to 4 loci and high for 5 to 7 loci). A: Correlation with ER/PR ...
Table 3.
Association of CpG Island Hypermethylation with Clinicopathological Features of Patients with Breast Cancer

This observation also supports the notion that concurrent hypermethylation of multiple CpG islands is commonly seen in breast cancer. 7,20 Several studies have described the existence of one or more methylator phenotypes, in which hypermethylation of multiple loci simultaneously occurs in various types of cancer. 21-23 Although the presence of a methylator phenotype could be attributed to a generalized deregulation of methylation control in the genome, our observation suggests that this event is not random in breast cancer; some promoter CpG island loci are seemingly more susceptible to this epigenetic alteration. 24 In this regard, we identified two loci, GPC3 and RASSF1A, which are concurrently hypermethylated in 40% of the breast tumors examined. Within these tumors, one subgroup (a) had additional methylation in HOXA5, WT1, and uPA loci, whereas the other subgroup (b) acquired frequent hypermethylation of 3OST3B, BRCA1, and DAPK1 genes (see Figure 5B [triangle] ). These methylation patterns seem to imply that DNA methylation may be progressively accumulated in different CpG island loci during tumor development. Future profiling of MTA data using various clustering algorithms will be useful in identifying different epigenetic pathways for molecular classification of breast cancer subtypes.

Concluding Remarks

We have demonstrated that MTA is a candidate gene approach for potential profiling of CpG island hypermethylation in hundreds of tissue genomes. This array-based method provides a new opportunity to assess clinicopathological significance of hypermethylated CpG islands in breast tumors and other types of cancer. Unlike other approaches, such as MSP or bisulfite sequencing, which focus on the evaluation of methylation details within individual CpG island loci, MTA is confined to uncover highly methylated CpG islands in multiple clinical samples. One advantage is that, like Southern blot, a single MTA nylon filter can be repeatedly used for probing with many CpG island loci, which allows for rapid assessment of potential methylation markers that may be clinically useful in predicting the treatment outcome of a cancer patient.

Acknowledgments

We thank Diane Peckham and Farahnaz Rahmatpanah for assistance in the preparation of the breast tumor samples.

Footnotes

Address reprint requests to Dr. Tim H.-M. Huang, Department of Pathology and Anatomical Sciences, Ellis Fischel Cancer Center, University of Missouri, 115 Business Loop I-70 West, Columbia, MO 65203. E-mail: .ude.iruossim.htlaeh@hgnauh

Supported by National Cancer Institute grants CA-69065, CA-84701, and CA-86305. C.-M. C. was a visiting fellow supported by the National Science Council, Taiwan (NSC39073F).

T. H.-M. H. is a consultant to Epigenomics, Inc.

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