By David N. Leff
What is a Denver boot? No, it's not a piece of fancy, hand-tooled cowboy footwear. A Denver boot is something like a giant padlock, clamped around the wheel of a car that's overstayed its parking welcome. An automobile wearing a Denver boot isn't going anywhere.
By a roughly similar token, if a stretch of chromosomal DNA wears methyl groups just upstream of a gene, that methylated gene isn't going anywhere.
Methylation is one of nature's ways of switching off a gene to keep it out of trouble.
It performs this trick by calling up methyl groups — three hydrogen atoms plugged into one carbon (CH3) — from the cell and attaching them to a cytosine-guanine base pair just above the target gene sequence. That puts said gene out of commission.
The molecular equivalent of the traffic cop who slaps on a Denver boot is the enzyme that transfers a methyl group to the gene. It's called methyltransferase.
Until recently, molecular biologists supposed there was only one methyltransferase, and only one form of methylation. They knew this ubiquitous process goes on day and night in all mammalian cells, from the embryonic to the adult.
They also knew the havoc that a defect in methylation could cause. Thus, in fragile X syndrome, a gene at the extreme tip of the male X chromosome carries an ever-elongating stretch of triplet nucleotide repeats — CGG (cytosine-guanine-guanine) — for which methyl groups have a special predilection (see BioWorld Today, Jan. 4, 1996, p. 1).
Linking methylation to cancer has been a tougher case to crack.
A recent clue was the discovery that in fact methylation comes not in one, but in two distinct persuasions — maintenance and de novo. Meaning that another methylation enzyme besides methyltransferase must be at work in the body. It hasn't been found yet, but its effects have.
"Methyltransferase," said molecular biologist Christoph Langauer, "is the maintenance enzyme. It guarantees that certain genes are always on, or off, as required; that the two double-helix strands of DNA will generate that same methylation pattern when they divide and make new DNA strands." Langauer is a research assistant in the molecular genetics laboratory of molecular biologist Bert Vogelstein, at the Johns Hopkins Oncology Center, in Baltimore.
He continued: "In certain circumstances, as for example fetal development, a new methylation starts somewhere de novo — where there had not been one before."
Researchers suspected that the same methyltransferase also honchoed these sudden new bouts of methylation. Then, only a few months ago, one laboratory deactivated that enzyme, and saw that de novo methylation kept right on happening without methyltransferase.
"It's one of those things," Langauer told BioWorld Today, "that the cell didn't experience before. So it reacts to this new stimulus with de novo methylation. And that leads to problems in certain types of cells."
Particularly, in cancer cells.
Protein Turned Blue; Ergo, Virus Did Infect
It led Langauer and his colleagues to a paper, of which he is lead author, in today's Proceedings of the National Academy of Sciences (PNAS), dated March 18, 1997. Its title: "DNA methylation and genetic instability in colorectal cancer cells."
He and his co-authors infected ten human colon cancer cell lines with a retroviral vector reconstructed from a mouse leukemia virus. Inside the viral envelope, it carried, along with some viral genes, marker genes for a protein that would turn blue when expressed.
"In general," Langauer explained, "when a cell gets infected with a retrovirus, it makes a kind of host-defense reaction against the foreign gene, to prevent its expression. That reaction takes the form of de novo methylation. It's sort of the same idea as the body's immune system, but at the DNA level rather than the cellular."
The team found two classes of reaction: cell lines that in the main don't express the transgene, and those that do — and turn blue.
"This was striking to us," Langauer recalled. "We didn't expect such a clear difference in the occurrence of de novo methylation. It's the major finding of our experiment."
What makes it new, he added, is how the Hopkins approach differed from current attempts by others. "They were actually comparing methylation patterns in tumor cells to the patterns of the same gene in normal cells. That doesn't really tell you a lot."
He explained: "The reason is, you really don't know what you should compare those cancerous cells to. You have a normal cell that at a certain stage becomes a tumor cell. When this happens to a cell — which it can at any stage — you don't know if it was methylated or non-methylated to begin with.
"What we have seen in our experiment," Langauer continued, "is that we get two classes, if you will, of colon cancer cells. One behaves as expected; that is, shows this de novo methylation of the retroviral genes. The second class does not, which is why we call it defective or aberrant de novo methylation. And we're sure it's new, because it has nothing to do with the old maintenance process."
Tumorigenesis: When Cells 'Inherit' Aberration
He pointed out that this defect persists as such cells divide and multiply. "So you'll find it in the daughter cells and their progeny and so on. It's the process of tumorigenesis."
"We are dealing with colon cells as a model," he said. "Most probably, it holds true for other types of tumor cells as well, though we didn't test that."
A different but perhaps related phenomenon separates Langauer's tumor cells into two either/or categories — those with aberrant de novo methylation, and those with defects in their genes that repair DNA nucleotide mismatches.
"We suggest," he said, "that this is definitely not a coincidence, but just might be a correlation between two different DNA pathways which play an important role in tumorigenesis. One way would be a defect in mismatch repair, which leads to inactivation of certain genes, which leads to tumors. The other has a defect in de novo methylation, which may fail to inactivate cancerous genes."
Pursuing this point, Langauer asked: "What's going on with these other colon cancers, which do not have mismatched repair defects? They constitute more than 75 percent of all inherited and non-inherited colon cancers. The interesting question now is: How is this aberrant de novo methylation related to cancer? We have some ideas, and that's what we're trying to figure out right now." *