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Home , DNA methylation

Proc. Natl. Acad. Sci. USA Vol. 94, pp. 2103–2105, March 1997

Commentary

Altered DNA and instability: A new pathway to ? Peter A. Jones and Mark L. Gonzalgo

University of Southern California͞Norris Comprehensive Cancer Center, Los Angeles, CA 90033

DNA methylation is a mechanism for changing the base The importance of this work is that it suggests an alternative sequence of DNA without altering its coding function. As a pathway for the inactivation of tumor suppressors. A weakness heritable, yet reversible, epigenetic change, it has the potential is that many of these studies have used data obtained from the of altering and has profound developmental analysis of a few sites whose relevance to and genetic consequences. The methylation reaction itself is gene control remains undetermined. This situation developed mechanistically complex and involves the flipping of the target because of difficulties in sequencing genomic DNA for 5-meth- out of the intact double helix, so that the transfer of ylcytosine distribution. In this regard, it is difficult to overstate the methyl group from S-adenosylmethionine can occur in a the contribution of Frommer et al. (14) who have simplified cleft in the enzyme (1). Cytosine methylation is inherently genomic sequencing by the use of bisulfite treatment, making mutagenic, which presumably has led to the 80% suppression the analysis of DNA methylation patterns a fairly routine task. of the CpG methyl acceptor site in eukaryotic organisms, The coupling of this precise and focused approach with the which methylate their . It contributes strongly to the nondirected genome scanning techniques such as that devel- generation of polymorphisms and germ-line mutations, and to oped by Ushijima et al. (5) and others (7–9) is likely to lead to transition mutations that inactivate tumor-suppressor genes rapid progress in this field. (2). Despite a 10- to 40-fold increase in the rate of transitions Almost all studies to date have focused on changes in at methylated versus unmethylated (3), methylation methylation status of endogenous genes in transformed cells is not only tolerated in several , but is actually relative to their presumed normal counterparts. In the other required for the of mammals (4). The related paper in this issue, Lengauer et al. (15) have taken a reasons 5-methylcytosine is essential for development remain different approach by introducing exogenous CpG-rich se- obscure, but most probably relate to the well documented quences in the form of retroviruses into colon cancer cell lines ability of methylation, particularly the methylation of CpG-rich and have come up with an entirely unexpected result. The promoters, to block transcriptional activation. Indeed, there is authors show that two distinct expression patterns were ob- growing evidence that methylation plays a pivotal role in key served in the transfectants. Five out of 10 lines failed to express developmental processes such as and the retrovirally encoded ␤-galactosidase gene, and these lines Ϫ stabilization of X-chromosome inactivation. It therefore is not were all deficient in DNA mismatch repair (MMR ). On the ϩ surprising that alterations in this essential epigenetic system other hand, cells competent for repair (MMR ) efficiently might play a role in (see Fig. 1 for examples). expressed the gene. Importantly, gene expression could be Ϫ Two papers in this issue of the Proceedings add new evidence induced in the MMR cells by subsequent treatment with to support the notion that alterations in the DNA methylation 5-azacytidine, a well known inhibitor of DNA methylation machinery are among the most common changes associated (16). This result, together with Southern blot analysis showing partial methylation of restriction enzyme sites in MMRϪ but with neoplasia and may have a causative role at an early stage ϩ in carcinogenesis. In their report, Ushijima et al. (5) have not MMR lines, suggests the existence of two distinct phe- notypes, i.e., MMRϪ, METϩ (where METϩ means methyl- modified the powerful representation difference analysis ϩ Ϫ (RDA) technique pioneered by Wigler and colleagues (6) to ation proficient) and MMR , MET . characterize and clone DNA fragments that show methylation The correlation between the MMR phenotype and expres- changes during murine hepatocarcinogenesis. This method sion is clear, but it will take some time to resolve the precise adds to the growing number of genome scanning approaches, role of the observed de novo methylation in . The authors confirmed the presence of methylation, using the including restriction landmark genomic scanning (7) and meth- powerful bisulfite genomic sequencing method discussed ear- ylation-sensitive arbitrarily primed PCR (8, 9), which all have lier, and observed a low, but measurable, level of methylation identified frequently altered methylation sites in cancer cells. in the viral long terminal repeat (LTR). The methylation The new methodologies are important in that they not only patterns were heterogeneous, and some LTR molecules were identify changes, but allow for the cloning of the relevant unmethylated, which might have contributed to the low levels fragments of DNA. The data from these nondirected ap- of expression seen in some of the transfectants. proaches all show that it is not simply the overall levels of DNA Both the Ushijima and Lengauer studies extend previous methylation that are altered in cancer, but rather that profound observations that methylation patterns are commonly altered changes occur in the distribution of methyl groups. in tumor cells, but do not address the question of how the de The above studies have been paralleled by approaches novo methylation of CpG islands occurs. De novo methylation focusing on the de novo methylation of the regions of CpG islands occurs in both endogenous sequences and of specific tumor suppressor genes. In several cases including retrovirally introduced LTRs during development (17). Un- Rb, , and VHL, the promoters are CpG rich and fulfill the fortunately, virtually nothing is known about how this is criteria for CpG islands, thus providing a rationale for believ- accomplished in normal or transformed cells, but it may well ing that they might be subject to methylation silencing (10–13). require the participation of an as-yet unknown de novo meth- ylase in addition to the copying or ‘‘maintenance’’ enzyme Copyright ᭧ 1997 by THE NATIONAL ACADEMY OF SCIENCES OF THE USA isolated and characterized by Bestor and colleagues (18). The 0027-8424͞97͞942103-3$2.00͞0 existence of such an enzyme seems almost a certainty, because PNAS is available online at http:͞͞www.pnas.org. ES cells with a complete knockout of the maintenance enzyme

2103 Downloaded by guest on September 26, 2021 2104 Commentary: Jones and Gonzalgo Proc. Natl. Acad. Sci. USA 94 (1997)

defect’’ (METϪ) directly facilitates the gain and loss of whole chromosomes leading to the genomic instability necessary for cancer development and progression. On the other hand, cells that are MMRϪ are hypothesized to have a normal methyl- ation proficiency and generate the required instability by the alternative pathway of mismatch repair deficiency. The hypothesis that DNA methylation and chromosomal integrity and segregation are linked has some experimental support. Feinberg et al. (22) found that colon tumors often have reduced 5-methylcytosine contents relative to normal tissues. They also observed that the three patients in their study with the highest levels of the modified base in their normal colonic epithelia all had Lynch syndrome (HNPCC). This observation, made before the discovery that HNPCC was due to the MMRϪ phenotype, suggests that the in vitro data obtained by Lengauer et al. (15) may indeed reflect the in vivo situation. Evidence that methylation may promote retention of chromosomes also has been provided by Hsieh (23), who found that methylated plasmids are maintained with higher efficien- cies than their unmethylated counterparts in transfected cells. However, other observations seem, at first sight, to be con- tradictory to this hypothesis. First, DNA hypermethylation (read de novo methylation) rather than hypomethylation pre- cedes allelic loss in neural tumors (24). Also, the elegant studies of Laird et al. (25) demonstrated that lowering the level of maintenance methylase by genetic or pharmacological means led to a significant decrease rather than an increase in the number of polyps in mice harboring the min mutation. Perhaps the difference lies in the of the ‘‘methylation defect’’ in the two systems. The methylation pattern present in a cell is likely to be determined by the interplay of a de novo methylase, a maintenance methylase, and possibly a demeth- ylase. The specific player is known in the transgenic experi- FIG. 1. Models for the role of 5-methylcytosine in cancer. [1] ments but unknown in the tumor systems. Until the relation- Signature mutations in the form of C 3 T transitions at methylated CpG sites are the hallmark of hydrolytic of 5-methylcy- ships between these activities are understood, it may not be tosine and commonly produce mutations in tumor suppressor genes possible to resolve this apparent discrepancy. Also, because such as p53. [2] Abnormal silencing of tumor suppressor genes can methylation presumably plays multiple roles in carcinogenesis occur through the epigenetic effects of DNA methylation at CpG (Fig. 1), it may be that different pathways are selected with islands, which may be localized to the promoters of growth regulatory differing degrees of penetration in diverse situations. genes such as p16, Rb, and VHL. [3] Induction of chromosomal The two new studies published today therefore add inter- instability may result from the inability of a cell to carry out de novo esting new dimensions to our understanding of the importance methylation as proposed in the report by Lengauer et al. (15). Circles of this essential epigenetic modification in cancer. They extend represent cytosines located at CpG dinucleotides; solid circles indicate methylated cytosines, open circles indicate unmethylated cytosines. the role of methylation in cancer beyond the fact that DNA T.S., ; PRO, promoter; LTR, long terminal methylation contributes directly to by once again repeat. pointing out the importance and prevalence of methylation changes, particularly de novo methylation in the initiation and still show residual methylation, including methylation of en- progression of cancer. Also, they raise intriguing questions as dogenous LTR sequences (19). An alternative route to altered to whether these common methylation defects contribute on a methylation in cancer cells may be mediated by disregulation more macro scale to the gross chromosomal imbalances that of the recently described activity (20). Further are the hallmark of all solid tumors. progress in understanding how methylation patterns are ac- quired and altered is obviously going to require a character- 1. Klimasauskas, S., Kumar, S., Roberts, R. J. & Cheng, X. (1994) ization of the basic enzymology for DNA modification. Cell 76, 357–369. What then is the relationship between the MMR and MET 2. Jones, P. A. (1996) Cancer Res. 56, 2463–2467. phenotypes? One highly plausible explanation would have 3. Yang, A. S., Gonzalgo, M. L., Zingg, J.-M., Millar, R. P., Buckley, been that unresolved mismatches created by the MMR defi- J. D. & Jones, P. A. (1996) J. Mol. Biol. 258, 240–250. 4. Li, E., Bestor, T. H. & Jaenisch, R. (1992) Cell 69, 915–926. ciency could serve as foci for de novo methylation. There is a 5. Ushijima, T., Morimura, K., Hosoya, Y., Okonogi, H., Tatem- strong rationale for this hypothesis, because Smith et al. (21) atsu, M., Sugimura, T. & Nagao, M. (1997) Proc. Natl. Acad. Sci. have clearly demonstrated that mismatches at CpG sites serve USA 94, 2284–2289. as highly efficient substrates for the human maintenance 6. Lisitsyn, N., Lisitsyn, N. & Wigler, M. (1993) Science 259, methylase. This does not, however, seem to be the case in the 946–951. colon cancer cell lines, because restoration of mismatch repair 7. Kawai, J., Hirose, K., Fushiki, S., Hirotsune, S., Ozawa, N., Hara, by transfer of the appropriate chromosome to the MMRϪ cells A., Hayashizaki, Y. & Watanabe, S. (1994) Mol. Cell. Biol. 14, did not abrogate their abilities to de novo methylate the viral 7421–7427. 8. Gonzalgo, M. L., Liang, G., Spruck, C. H., Zingg, J.-M., Rideout, et al. LTR. Lengauer (15) come up with an ingenious alter- W. M. & Jones, P. A. (1997) Cancer Res. 57, 594–599. native explanation to account for their observations and 9. Huang, T. H.-M., Laux, D. E., Hamlin, B. C., Tran, P., Tran, H. explain the generation of genomic instability in the 85% of & Lubahn, D.-B. (1997) Cancer Res. in press. colon that have an MMRϩ, yet METϪ, phenotype. 10. Greger, V., Debus, N., Lohmann, D., Hopping, W., Passarge, E. They propose that the as-yet uncharacterized ‘‘methylation & Horsthemke, B. (1994) Hum. Genet. 94, 491–496. Downloaded by guest on September 26, 2021 Commentary: Jones and Gonzalgo Proc. Natl. Acad. Sci. USA 94 (1997) 2105

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