A Publication of the James Buchanan Brady Urological Institute Johns Hopkins Medical Institutions

Chaos in the Chromosomes, and New Keys to Advanced Cancer

The more vicious the prostate cancer, the crazier this chromosomal mix-up becomes.

Of all the bizarre things that can happen in the genetic structure of men with prostate cancer, this may be one of the most bizarre-unstable chromosomes, which break apart and patch themselves together with completely different chromosomes. Groundbreaking genetic work at the Brady Urological Institute, building on decades of pioneering research by a handful of scientists including Don Coffey, William Isaacs, William Nelson, Alan Partin and others, has shown that the more vicious the prostate cancer, the crazier this chromosomal mix-up becomes.

Coffey, whose legendary work on the architecture of cancer cells laid the foundation for this most recent discovery, thinks he knows what makes this happen, and he even has two key culprits-and maybe, two new pathways for blocking the very worst kinds of prostate cancer.

In this case, the architecture of cancer cells is a bit different from the structural landmarks pathologists use to determine the Gleason grade and stage of cancer. If we were talking about buildings-say, a medieval cathedral-what the pathologists make note of would be things like apses, arches, naves, and buttresses. What Coffey and colleagues are investigating would be the stones and mortar.

Like a Funhouse Mirror
To backtrack two and a half decades: Coffey helped create a new subspecialty of cell biology when he discovered how DNA- the body's genetic material-is organized within a cell's nucleus. Scientists had long known that the nucleus of a cancer cell is odd-looking; it's distorted, like someone's reflection in a funhouse mirror. When this happens, the chromosomes often lose their normal shapes, as well. Coffey discovered that there is a scaffolding inside each nucleus, a skeleton that determines its shape. He called this the nuclear matrix. He found that the DNA was attached to this matrix in countless loops (actually, about 50,000 little loops), each locked at its base to the scaffolding. Coffey and colleagues also showed that those loops are a genetic hot spot, where DNA replicates itself. They named the loops "replicons," and found that DNA reproduces itself in little pieces-the loops-instead of as one very long string of information.

Several years ago, William Nelson discovered a key enzyme, called topoisomerase II (topo II for short) at the base of those loops, and Alan Partin found that the composition of the nuclear matrix was different in normal and in cancer cells. Then Coffey and colleagues noticed that in cancer cells, the loops were unwinding -because topo II was unwinding them. Another strange thing: The composition of the proteins in the nucleus was changing-and another scientific subspecialty, called proteomics, the study of proteins expressed by various genes, was born.

Coffey likens what's happening here to the working of an audio cassette player. "The tape is the DNA, the cassette is the nucleus, and the cell is the whole tape recorder," he explains. "The messenger RNA is the electricity coming off the tape, and the protein is the sound the tape recorder is making. The transient material is the electricity, and the product is sound. What we want to know is the sound that's coming off the tape recorder-what protein is the cell making? That pattern is proteomics." There's also plenty of static. "About 95 percent of the DNA in a chromosome never makes sound, or protein. We used to call it junk DNA,' but now we find it's this silent, repetitive DNA that makes up the distance between letters, words, and paragraphs." (Some of these spaces are now called introns.)

A Bad Patchwork Quilt 
At the ends of the chromosomes are little pieces of repetitive DNA-small caps, like the aglet at the tip of a shoelace-called telomeres. "If you look at your shoe lace, as long as the aglet's there, it's pretty stable," says Coffey. "But when the aglet wears away, the lace starts fraying, and the chromosome starts getting in trouble. When that happens, the cell is unhappy; the DNA is not doing well."

Losing the telomeres is one cause of the spooky chromosomal rearrangement. Another culprit is a protein at the base of those loops on the matrix, with the alphabet- soup name of HMGI(Y). "It's like a railroad switch that can make the train go down a different track," says Coffey. "The train is attached to the matrix, the railroad track, but this little protein can flip the switch, and get the trains mixed up." HMGI(Y)'s close buddy, genetically speaking, is topo II, and they work together. HMGI(Y) puts the railroad tracks in a dangerous situation, and topo II is the miscreant hammering the boards together.

One suspicious fact about HMGI(Y) is that it's not found in normal adult cells. It's usually a part of embryonic cells, but as we mature, it disappears. It reappears in cancer. "So cancer is a lot like developmental tissue," notes Coffey, "and it picks up a lot of embryonic properties-like a stem cell for the cancer." Coincidentally, telomerase (which makes telomeres) is also in stem cells, and in cancer cells, too.

When HMGI(Y) is added to DNA, it causes it to form four-way junctions (think of any awful exit of I-95)-and makes "a perfect place for DNA rearrangement to occur," says Coffey. "HMGI(Y) is in the right position at the base of those loops, it can form a four-way junction, and we know that topo II is sitting there, flipping the DNA open and closed. It's the ideal place to mess it up, especially if the matrix isn't normal. So rather than two train tracks going left and right, they now cross over, and the cars begin to pile up."

Coffey and colleagues have a way of painting the chromosomes to make each appear as a different color under a microscope. "Imagine yourself standing in front of a white wall, and you pick up a brush and paint the first chromosome as a red stripe, all the way down the wall," explains Coffey. "The longest by size is chromosome 1; the smallest is number 22. Next, you paint the 2 chromosome blue, and make it a little shorter. By the time you get to the end of the wall, you have a picket fence that goes down, and gets shorter and shorter, 22 different colors." When the scientists painted the chromosomes of prostate cancer cells in this way, they were shocked at the result. "Holy mackerel! There's a piece of red on top of a piece of blue, with a piece of green at the bottom. Or a piece of yellow, a piece of blue, and a piece of green. Then two that look okay, but the fence has too many pickets-instead of 22, it's got 37. This thing is messed up."

The scientists looked for this crazy patchwork effect in three well-known strains of prostate cancer. Like the porridge that Goldilocks taste-tested, two are extreme- one notoriously vicious, (Coffey calls it a "mean sucker that grows like crazy"), one pretty mild, and one right in the middle. In rat tumors, the worse the cancer, the more metastatic and aggressive it is-the more HMGI(Y) it has. In humans, the higher the Gleason grade, the more HMGI(Y) there is. In Coffey's recent experiments to study chromosomal rearrangement, the most benign cell line had some rearrangement, but-this is important-it was balanced rearrangement. "If you took a deck of cards, and put the top half of the two red queens on the bottom half of the two black jacks, you would see two other new cards made, with the bottom half of the red queens and the top half of the black jacks." This is called a "reciprocal translocation," and it is balanced.

Coffey thinks he knows what makes this happen, and he even has two key culprits-and maybe, two new pathways for blocking the very worst kinds of prostate cancer.

But the very worst cell line resembled a train wreck-"31 unbalanced chromosomes. It looked like a trash dump, all parts combined in unbalanced translocations, colors mixed up in every way you could imagine."

When the Brady scientists inserted a piece of HMGI(Y) into the tamest cancer, the one with the balanced rearrangements, it became unhinged-and made the unbalanced matches found in the worst form of cancer. The extent of the damage matched the level of HMGI(Y).

Now, what to make of all this? "We can cure some cancers-such as leukemias, lymphomas-easily," says Coffey. "They are the kinds of cancers that have balanced rearrangements of chromosomes." One such cancer, Burkett's lymphoma, has an "8-14 translocation"-an even mix-andmatch of chromosomes 8 and 14. This form of cancer affects children in Africa, and it's horrible: "The whole face is distorted, like the most severe mumps ever," says Coffey. "It looks like the person has elephantitis of the face; the lymph glands are growing like a house afire. But if you treat this person with cytoxan, he goes home. He's cured!" Another example is advanced testicular cancer, which struck American cyclist Lance Armstrong. "Some of these men have so many metastases in their lungs, it looks like they're drowning in tumor; they're spitting up blood, they can't get enough oxygen. Lance Armstrong had three metastases in his brain the size of golf balls. But he was treated, his cancer went away, and he wins the Tour de France. I want to know why that's so."

Coffey's latest research has found that the cancers most difficult to cure are the kinds-like prostate cancer-prone to unbalanced rearrangements. "We know that the reason it's so hard to cure advanced prostate cancer is because of its genetic instability. It spins off such a variety of cells that it's almost impossible to beat it." The latest piece of the puzzle, the subject of some of the cutting-edge research at the Brady right now, is the telomeres- the tips of the shoelaces, or chromosomes. Knocking out telomerase-getting rid of the telomeres-causes unbalanced chromosomal rearrangement, too. "So we've got two ways to cause unbalanced rearrangement-HMGI(Y), and losing telomerase. Both of these can happen in a man with prostate cancer. Can we make a target out of this? Maybe we can go after HMGI(Y) and telomerase, and somehow stop this mismatched rearrangement. Currently, we're trying to figure out the best way to take this to the patient."


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