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Fig. 3.

Hypermethylation and loss of expression of the Versican gene in colorectal cancer. A, map of theVersican gene first exon (filled box) and flanking regions. Top, CpG sites. Arrows, location of the primers used for bisulfite-PCR. Methylated alleles are digested to 162 and 37 bp by the restriction enzyme TaqI. Unmethylated alleles remain undigested because of the absence of this site after bisulfite conversion of unmethylated cytosine. B, Versican methylation in colorectal cancer and normal colon mucosa. Bisulfite-treated DNA was amplified using primers V1 and V2. The PCR product (199 bp) was digested with TaqI and electrophoresed in a 3% agarose gel. TaqI cleaves only the methylated allele, yielding bands at 162 and 37 bp (the 37 bp is not visible by agarose gel electrophoresis). Hypermethylation of the Versican gene was detected in most of the colorectal cancer cell lines tested (top) and 70% of primary colorectal cancer (T1, T2, and T4–6; middle panel). N, normal colon; T, colon tumor. Methylation of Versican was also detectable in normal colon mucosa and progressively increased with age (bottom panel, all samples are from normal colon mucosa; age of the patient is indicated on top of each lane). Percentage Versican methylation (shown at the bottom of each lane) was determined by comparing the intensity of the bands derived from the methylated (162 bp) and unmethylated (199 bp) alleles. C, age-related methylation of Versican in colonic tissues. Columns, average percentages of methylation for each decade; bars, SEM. The number of samples examined for each decade is 4, 6, 3, 6, 4, 9, 10, and 4, respectively. D, expression ofVersican (V) in colonic tissues. Versican expression was determined by RT-PCR in normal colon (left), hemimethylated cell lines (HCT116 and HT29), highly methylated cell lines (LOVO, SW48, SW837, RKO, CEM, RAJI, DLD1, Caco2, and SW480), and colorectal cancer cell lines treated with the methylation inhibitor 5-aza-2′-deoxycytidine (tr). All reactions were controlled by RT (−) samples (data not shown). The cDNAs were also amplified using glyceraldehyde-3-phosphate dehydrogenase primers (G) to verify RNA integrity. Sizes of the PCR products are 292 bp for Versican and 306 bp for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Note that all of the highly methylated cell lines have very low levels of expression (compared with normal colon), and that this expression is significantly increased by treatment with the methylation inhibitor (right).

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Discussion

MCA is a novel PCR-based technique that allows for the rapid enrichment of hypermethylated CG-rich sequences, with a high representation of methylated CpG islands. This technique can have several potential applications. MCA can be useful for the determination of the methylation status of a large number of samples at multiple loci relatively rapidly. By optimizing the PCR conditions, it should be readily adaptable to the study of the methylation status of any gene that has two closely spaced SmaI sites. As shown here, there is a very high concordance rate between MCA and other methods for the detection of hypermethylation, such as Southern blot analysis and bisulfite-based methods. However, compared with other methods, MCA has some disadvantages in that: (a) it requires good quality DNA, excluding the study of paraffin-embedded samples; (b) it examines only a limited number of CpG sites within a CpG island; and (c) it is sensitive to incomplete digestion using the methylation-sensitive enzyme SmaI. Nevertheless, many steps in MCA are amenable to automation and, by allowing for the examination of multiple genes relatively quickly, it may have important applications in population-based studies of CpG island methylation.

An important application of MCA is in the discovery of a novel gene hypermethylated in cancer. As demonstrated here, MCA coupled with RDA is a rapid and powerful technology for this purpose and compares favorably with other described techniques (13,14, 15, 16) . In addition to the identification of genes hypermethylated in cancer, MCA could potentially be used to discover novel imprinted genes using parthenogenetic DNA, as well as novel X chromosome genes.

Using MCA/RDA, 33 differentially methylated clones were identified and characterized in detail. By sequencing, we found that 29 of the 33 clones satisfy the criteria of CpG islands, demonstrating that MCA can represent CpG islands specifically. Of these 29 clones, 6 were already known genes (PAX6, Versican, α-tubulin, CSX, OPT homologue, and rRNA gene).

Of the known genes recovered in this study, Versican is most interesting in that this proteoglycan is an RB1-inducible gene (24) , suggesting that down-regulation of this gene product may have an important role in colorectal carcinogenesis, where RB mutations are rare. Although the relation between Versican and cancer was first described for hypomethylation of this gene involving a nonpromoter region (25) , our data clearly show that aberrant methylation of the Versican gene promoter is correlated with silencing of this gene. Here, we also show that promoter methylation of Versican is associated with aging in normal colon mucosa. This is similar to other genes reported to be methylated as a function of age in normal colon, including ER (5) , IGF2 (26) , MyoD, and N33 (27) . Although the functional significance of age-related methylation is still not clear, we have suggested that it constitutes a type of field defect in the colon, which partially explains the dramatic age-related increase in colorectal cancer incidence (5) .

Methylation of the CSX and OPT genes does not coincide with their 5′ end and is therefore not expected to silence these genes. It is possible, however, that these CpG islands are associated with alternate transcripts of the genes or with other nearby genes, which would then be silenced by methylation (28) . Nevertheless, these data confirm previous studies showing that hypermethylation in cancer does not always involve promoter-associated CpG islands (29) . PAX6 is a gene involved in eye development that, interestingly, was also identified as differentially methylated in cancer using arbitrarily-primed-PCR (29) . In that study, the CpG island recovered was in the coding region of the gene, whereas MINT8 corresponds to a PAX6 enhancer present in the 5′ region of the gene. Methylation of the PAX6 enhancer is likely to influence its transcription. However, it is not known whether this gene is expressed in normal colonic tissues. Finally, methylation of ribosomal genes has been seen previously in aging tissues (30) and therefore is not surprising to find in cancers.

The significance of methylation of the other clones obtained in this report is not clear. The methylation of some of these may simply reflect the global redistribution of 5-methylcytosine during cancer development (6 , 7) with little functional significance. However, because some of the clones recovered are in the exon 1 region of expressed genes, identification of new tumor suppressor genes might be facilitated by using MCA/RDA clones as probes for screening cDNA libraries. Indeed, on the basis of their chromosome location, several clones map to chromosomal regions thought to harbor tumor suppressor genes because they are highly deleted in various tumors (e.g., chromosomes 1p35, 3p25–26, 7q31, 17q21, and 22q11–ter).

In conclusion, we have developed MCA, a novel method to selectively amplify methylated CpG islands. Using MCA coupled with RDA, 33 clones differentially methylated in colorectal cancer were isolated, including several known genes. These clones will be useful markers to identify novel genes silenced by hypermethylation in cancer and may also be useful markers for early detection and prediction of prognosis in colorectal cancer.

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Acknowledgments

We thank the staff at the Johns Hopkins Core Sequencing Facility for excellent technical assistance.

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Footnotes

• 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.

• ↵1 This work was supported by National Cancer Institute Grants CA77045 and CA54396 and Colon Cancer Spore Grant CA62924 from the NIH. M. T. is a postdoctoral fellow from Japan Society for the Promotion of Science. N. A. is supported by NIH training Grant 1-T32-DK07713. J-P. J. I. is a Kimmel Foundation Scholar.

• ↵2 To whom requests for reprints should be addressed, at The Johns Hopkins Oncology Center, 424 North Bond Street, Baltimore, MD 21231. Phone: (410) 955-8506; Fax: (410) 614-9884; E-mail: jpissa@welchlink.welch.jhu.edu.

• ↵3 The abbreviations used are: MCA, methylated CpG island amplification; RDA, representational difference analysis; RT-PCR, reverse transcription-PCR; MINT, methylated in tumors.

• Received February 4, 1999.

• Accepted March 30, 1999.

• ©1999 American Association for Cancer Research.

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