DNA methylation, the driving force behind genetic imprinting and X-chromosome inactivation, is also crucial during the earliest stages of embryogenesis. But over the past 20 years, research has focused on DNA methylation gone awry; it seems that both global hypomethylation of the genome and hypermethylation of tumor suppressor genes may play roles in carcinogenesis.
Too Little
Hypomethylation in cancer was first seen in the early 1980s by Melanie Ehrlich, Ph.D., now professor at Tulane Universitys Cancer Center, New Orleans.
"The cancers were often deficient in global DNA methylation levels, compared with the range [of methylation] found in normal human tissues," she said.
One of hypomethylations effects, said Ehrlich, is to increase karyotypic instability, a phenomenon seen in ICF syndrome (immunodeficiency, centromeric region instability, and facial anomalies), the only known disease involving inheritance of abnormal DNA methylation. The syndrome involves mutations in the gene for one of the three known DNA methyltransferases.
Global hypomethylation is not a universal hallmark of cancer, however. "Some tumors show global demethylation, and others do not," said Timothy Bestor, Ph.D., associate professor of genetics and development at Columbia University, New York.
Too Much
Hypermethylation of tumor suppressor genes has been demonstrated in several human cancers. BRCA1, E-cadherin, p16, DAP-kinase, hMLH-1, and other tumor suppressor genes have been studied in cancers including those of the breast, ovary, head and neck, and endometrium. Hypermethylation of specific tumor suppressor genes has been found in 10% to 60% of tumors studied.
The retinoic acid receptor-ß2 is one example. Several studies have shown that expression of RAR-ß2, believed to be a tumor supressor gene, is repressed in breast cancer cell lines. A study in this issue of the Journal by Martin Widschwendter, M.D., of the University of Innsbruck, Austria, and colleagues (see article, p. 826) found that methylation the RAR-ß2 gene may be the culprit.
Other researchers are also exploring the role of methylation in tumor suppressor gene inactivation.
"Weve been describing [hypermethylation] as an alternative to mutational activation," said James G. Herman, M.D., assistant professor of oncology at the Johns Hopkins Oncology Center, Baltimore. "Just like a gene thats inactivated by a mutationespecially a tumor suppressorthere are epigenetic events that can substitute for mutation."
Human DNA methylation can occur only at a cytosine base immediately upstream from a guanine basea conformation known as a CpG dinucleotide. The promoter regions of more than half of all human genes contain stretches of CpG-rich DNA, known as "CpG islands."
During methylation, cytosine is rotated out of the DNA double helix by a DNA methyltransferase, which then "tags" the cytosine with a methyl (CH3) group before returning it to the helix. Research indicates that a methyl-binding protein then binds to the methylated CpG units, attracting other enzymes and proteins to form a complex that prevents transcription from occurring.
In the past year, Herman and colleagues have published research showing hypermethylation at specific genes in head and neck cancer, endometrial cancer, and B-cell malignancies. For example, late last year, the group found that endometrial tumors and endometrial hyperplasias showing microsatellite instability (MSI)the addition of new pieces of DNA with repeating nucleotide segmentsalso were likely to have methylated MLH-1 genes, while those tissues without MSI did not.
The work "demonstrates that its the loss of MLH-1 thats causing microsatellite instability," said Herman. "MLH-1 is part of the repair machineryif you dont have that protein, you dont have mistakes repaired, which leads to other mutations."
Peter A. Jones, Ph.D., D.Sc., distinguished professor of medicine and director of the University of Southern Californias Norris Comprehensive Cancer Center, said that solid research supports hypermethylation as a factor in some cancers.
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As for the studies that find tumor suppressor hypermethylation in only a fraction of tumors, "I could say the same thing for any gene," he said. "Take the p53 genein certain cancers its mutated 10% of the time. Does that mean its not important?"
Bestor has doubts. "The lack of a plausible mechanism for de novo methylation of tumor suppressor genes makes me skeptical . . . . I cant say that methylation cannot be involved in carcinogenesis, only that a number of questions must be answered before that conclusion can be drawn."
Bestor said that more than 90% of the methylated bases in the genome are within transposonssmall, mobile sequences in DNA that can insert copies of themselves at random sites within chromosomes. "The instability of methylation patterns in tumor cells is likely to reflect a conflict between the transposons and the host genome, in ways that are not yet clear," he said.
Role of Methyltransferase
In 1995, researchers at the Whitehead Institute for Biomedical Research at MIT crossed familial adenomatous polyposis (FAP) micepredisposed to develop colon tumorswith mice that produced a shortage of DNA methyltransferase. They hypothesized that the offsprings decreased methylation ability would lead to increased tumors in the offspring.
However, the resulting mice actually had 60% fewer tumors than did controls. The authors wrote, "DNA methyltransferase activity contributes substantially to tumor development in this mouse model of intestinal neoplasia."
How DNA methyltransferase might contribute to carcinogenesis remains largely unclear, though one possible conclusion is that increased DNA methyltransferase leads to the inactivation of genes that normally suppress carcinogenic pathways. Some studies have shown higher levels of DNA methyltransferase in tumor cells compared with normal cells, but others have shown only small increases or no differences at all.
Moshe Szyf, Ph.D., associate professor of pharmacology at McGill University Medical School, Montreal, hypothesizes that DNA methyltransferase disrupts the cell cycle, pushing damaged cells to replicate when they should undergo apoptosis and die.
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Methylgene Inc., a Montreal-based company, entered MG98an antisense drug targeting DNA methyltransferase in phase I clinical trials last year. The National Cancer Institute of Canada Clinical Trials Group in Kingston, Ontario, and four cancer centers in North America are also involved.
Cant See the Forest for the CH3s
Alan Wolffe, Ph.D., vice president and chief scientific officer at Sangamo Biosciences Inc., Richmond, Calif., believes that methylation is only one piece of a larger puzzle. "My opinion is that the epigenomethe way the genome is packagedis key to understanding development, differentiation, embryogenesis, and tumorigenesis," he said.
Wolffe spent the past decade directing the Department of Molecular Embryology at the National Institute for Child Health and Human Development, where he and colleagues came to understand the complexity of "the biochemistry of how differentiation happens and how genes are silenced. There are many bells and whistles built into this."
Jones also takes a "big picture" approach.
"Im not sure we should focus on hyper[methylation] or hypo[methylation]," he said. "Everyone focuses on their own little thing without looking at the whole picture. You cant just use the word methylationyou have to know where it is. Very often there is this simple seesaw phenomenonthe pattern changes, just as a seesaw goes up and down."
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