NEWS

For Telomeres, Longer Is Not Always Better

Karyn Hede

"Short people got no reason to live," goes the Randy Newman song. The same used to be thought of cells with short telomeres, those indispensable nubs of repetitive DNA and protein coating that protect the ends of chromosomes. But if Randy Newman was singing about telomeres, he would have to rewrite his lyrics. New findings are overturning what scientists thought they knew about the importance of telomere length; the role of telomerase, the enzyme that maintains telomeres; and how cancer cells might respond to anti-telomerase treatment.

Nobel prize–winning geneticist Barbara McClintock, Ph.D., first posited that telomeres protect chromosomes from the assaults of exonucleases, as well as from fusions and recombinations that can become the fate of uncapped chromosomes. Such genetic damage usually leads to cell death, but occasionally cells escape death and instead become cancerous.

Certain cells, including cancer cells, protect their telomeres from further trimming as the cells multiply by activating telomerase, the enzyme that lengthens and maintains telomere caps. When scientists initially looked for telomerase in normal mammalian cells, they found it only in the gamete-producing cells of the gonads and some stem cells of the bone marrow. This seemingly clean-cut dichotomy in telomerase activity has excited cancer researchers, who see telomerase as a potential target for new anticancer therapies.

But as so often happens in science, what once seemed simple has turned out to be complex.

"The black and white world is eroding away and becoming sort of gray," said Elizabeth Blackburn, Ph.D., a biochemist at the University of California at San Francisco and a co-discoverer of telomerase. "Now it’s turning out that ... in various cells of the proliferating kind—renewing cells of the intestines and hair follicles—people are clearly seeing telomerase is turned on."



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Dr. Elizabeth Blackburn

 
In fact, a study published in Cell last summer showed that telomerase is turned on in normal human fibroblasts, a finding that contradicts previous results. William Hahn, M.D., Ph.D., of Dana-Farber Cancer Institute, Boston, and Robert Weinberg, Ph.D., and Sheila Stewart, Ph.D., of the Whitehead Institute for Biomedical Research, Cambridge, Mass., and their colleagues discovered a low but reproducible level of telomerase in fibroblasts during DNA replication by using an extremely sensitive antibody and artificially synchronizing the cells’ division. What’s more, the researchers discovered that telomerase plays a crucial role in maintaining the very tip of the telomere, even as the overall telomere length shortens.



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Telomeres, those indispensable nubs of repetitive DNA and protein coating, protect the ends of chromosomes. The role of telomere length and telomerase—the enzyme that maintains telomeres—in cancer cells has turned out to be a complex research question. (Image courtesy of the U.S. Department of Energy Human Genome Program, http://www.ornl.gov/hgmis)

 
"What we are discovering is that the overall length is not really that important in triggering senescence, but that instead what is important is the configuration of the single-stranded overhang [at the very tip of the telomere] and the associated proteins," said Weinberg. "If this single-stranded overhang becomes damaged, even in a telomere that is otherwise very long, that damage at the tip will provoke senescence."

Instead, loss of the single-stranded overhang appears to trigger the cells’ DNA repair pathways so that the damaged ends fuse with other chromosomes, creating the very chromosomal rearrangements that can lead to cell death or, in some cases, to cancer. But just how chromosome fusions are initiated has been a matter of some debate.

Fusion or Confusion

Carol Greider, Ph.D., and her colleagues at Johns Hopkins University, Baltimore, have created something of a stir by saying they have evidence that overturns a 60-year-old theory about how short telomeres lead to genomic instability.

Greider, Blackburn’s collaborator in the telomerase discovery, and Jennifer Hackett, Ph.D., now at Harvard, reported in December in the journal Molecular and Cellular Biology that short yeast telomeres are first attacked by exonucleases that chew away at the chromosome ends before they fuse with other chromosomes in the cell’s ill-fated attempt to repair the genetic damage. This differs from the accepted dogma, first espoused by McClintock, which says that cells interpret chromosomes with short telomeres as being broken and activate their DNA repair machinery to fix the damage by fusing the short telomere to another chromosome tip. These fused chromosomes can be pulled to opposite poles when the cell divides and torn apart, beginning a cycle called the "breakage-fusion-bridge" pathway, which can lead to cancer.

In their experiments, Hackett and Greider inserted genetic markers into a yeast chromosome so they could trace which genes are lost first when telomeres get too short. Instead of random damage, as would be expected if breakage occurs randomly, they observed that the markers near the tip of the chromosome were most likely to be lost. When they repeated the experiments in a strain of yeast missing a key exonuclease, they found that there were fewer chromosome rearrangements when the exonuclease was missing.

"Nonreciprocal translocation is one of the most prevalent rearrangements that [is] seen in human cancer cells," said Greider. "The structure that we found in the yeast is the same structure that you see in human cancer cells. From what I’ve seen, the real fundamental aspects of telomere biology are extremely conserved. So my guess is there will probably be a similar kind of mechanism in human cells."

Greider said that the findings have wide-ranging implications. "If you’re looking at what’s going to be the proximal change in cells [leading to chromosome instability] knowing the initial mechanism is paramount. This is a fundamental change in thinking at a very basic level."

But others are not yet convinced that the Greider team’s findings in yeast will translate to human cancer.

"I believe that degradation is not a major mechanism for instability due to telomere loss in mammalian cells," said John Murnane, Ph.D., professor of radiation oncology at UCSF.

Murnane and his colleagues reported in the July 2002 issue of Molecular and Cellular Biology that, in mouse embryonic stem cells, sister chromatid fusion results when telomeres are lost at chromosome ends. The findings are consistent with the theory that breakage-fusion-bridge cycles are the major cause of chromosome instability when telomeres are short.

He said that, although exonucleases may be able to chew into coding regions in yeast, in mammalian cells, there is too much noncoding DNA at the ends of chromosomes for exonucleases to be much of a factor.

"In view of results we have, I tend to think degradation doesn’t play as significant a role in mammalian cells," he said.

In the Clinic

Whereas the intricacies of telomere function have yet to be fully worked out, clinical application of anti-telomerase therapies for cancer continues full speed ahead.

One of the most fully developed strategies relies on the observation that telomerase is overexpressed in nearly all cancer cells, making it a natural target for strategies that try to train the immune system to attack cancer cells.

"Telomerase is quite attractive to us because it is a so-called universal tumor antigen," said Johannes Vieweg, M.D., a Duke University Medical Center immunologist who is leading an immunotherapy clinical trial in patients with metastatic prostate cancer. Vieweg and Duke immunotherapy pioneer Eli Gilboa, Ph.D., are priming potent antigen-presenting dendritic cells with telomerase RNA in an attempt to get the body’s own immune system to attack cancer cells. A phase I safety trial, which enrolled 18 patients, is complete, and Vieweg presented preliminary results at the American Society of Hematology (ASH) annual meeting in San Diego in December. At the meeting he said all patients but one had strong telomerase-specific cellular immune responses and that no patients had treatment-related side effects. This initial study was designed to study patient safety and dosing schemes, and Vieweg said preliminary clinical results are promising.

"We have seen an impact on serum PSA [prostate-specific antigen] that suggests a slowdown of tumor progression," he said. "... That tells me we might be on the right way."

In a similar strategy, Gustav Gaudernack, Ph.D., and his colleagues at the Norwegian Radium Hospital in Oslo, Norway, are using peptide fragments of the telomerase protein in an injectable vaccine designed to stimulate an immune response to cancer cells. The scientists reported at the 2003 American Society of Clinical Oncology annual meeting in Chicago in June that patients with advanced prostate cancer (47 patients), melanoma (10 patients), and non–small-cell lung cancer (20 patients) received the vaccine over 10 weeks with some patients receiving monthly booster shots thereafter. Gaudernack said that 80% of the intermediate-dose patients in the prostate cancer study had measurable immune responses. In addition, he said that none of the patients in any study have had adverse effects from the vaccine.

"It is fair to conclude that targeting telomerase through hTERT [telomerase] vaccination is a promising strategy to combating cancer," said Gaudernack. "The best way to vaccinate and the optimal clinical setting for testing immunotherapy against hTERT will hopefully be determined in the next 1 or 2 years."

However, laboratory findings that telomerase is present in more normal, actively dividing cells than previously thought may make anti-telomerase cancer strategies problematic.

"Finding telomerase’s presence in normal cells suggests that normal cells may be susceptible to anti-telomerase therapies," said Weinberg. "Therefore, the therapeutic index, i.e., the selective effect on cancer cells that was previously predicted, may not be realized."

Blackburn, meanwhile, has recently shown that introducing a complementary antisense oligonucleotide fragment to telomerase RNA can induce a rapid apoptotic cell death response in cancer cells, even if the cell’s telomeres are still long.

"This is really new," said Blackburn. "Things may be a lot better than we thought originally. What this is saying is that telomerase is clearly playing an important role in the continuing life of the cancer cell, not just in keeping the telomeres long."

The discovery calls into question the role of telomere length and telomerase in triggering cell death and the role that telomerase plays in maintaining cancer cells.

"The basic story is there," said Blackburn. "Telomerase is doing lots of other things besides making telomeres long."



             
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