Science, Feb 12, 1999 v283 i5404 p947
Titia de Lange; Ronald A. DePinho
If oncogene activation is akin to a jammed gas pedal and tumor suppressor inactivation to loss of the brakes, what then is telomerase activation in this hackneyed cancer-car analogy? Telomerase, the reverse transcriptase that maintains the ends of eukaryotic chromosomes, has long been stigmatized for its association with human cancer. This judgment has grown from the observations that, although most normal cells are devoid of telomerase’s enzymatic activity and lack its main protein component (human telomerase reverse transcriptase, or hTERT), nearly all human tumors express hTERT and have active telomerase (1, 2). New data confirm that telomerase is neither tumor suppressor nor oncogene, underscoring the unique role of telomeres in tumorigenesis.
In normal cells, insufficient telomerase activity and a finite store of telomeric DNA limit the number of divisions a cell can undergo before critical telomere shortening signals entry into replicative senescence, defined by a finite capacity for cell division (3, 4). As such, replicative senescence is believed to represent a prominent genetic roadblock on the way to cancer, one that can be avoided by activation of telomerase (4, 5) but that can also be detoured by oncogene activation (for example, Myc) or loss of tumor suppressor function (such as RB/p53) (6-8).
However, cells that use Myc or loss of RB/p53 to circumvent senescence will eventually experience rampant genome instability due to the loss of their telomeres and require a mechanism to maintain these essential elements (9). Thus, no matter which road the aspiring cancer cell travels, counteracting telomere shortening appears to be a key step. On the basis of the frequent activation of telomerase in human cancer, upregulation of this enzyme is apparently the simplest way toward this end.
Thus, telomere erosion and the associated limitations in replicative life-span have been proposed as a potent tumor suppression mechanism, and telomerase was censured as a “bad” enzyme whose activity subverts our normally constrained somatic cells by providing the opportunity for boundless growth. Initial efforts to understand the mechanics underlying the telomerase-tumorigenesis connection seemed to yield more questions than answers. Is its activation essential for cancers to develop? Is telomerase really all that is needed for cellular immortalization, and will enforced somatic expression of telomerase lead to a cancer-prone condition?
Definitive answers to these questions have yet to emerge. However, the first major advance was provided a year ago with the finding that ectopic expression of hTERT in primary human cells could confer endless growth in culture (4, 5). Although the cells in question, human foreskin fibroblasts and retinal pigment epithelial cells (RPEs), normally ceased dividing after 40 to 80 population doublings, telomerase-positive derivatives able to maintain their telomeres progressed unimpeded beyond that usual lifespan and have now been maintained in continuous growth for more than a year (10, 11). For practical purposes, these cells can be viewed as immortal—a characteristic illegitimately appropriated by many human cancers but normally preserved for the few cells that make up our germ line.
These studies received much attention as a potential cellular fountain of youth, with visions of an immediate impact on normal tissue and transplant repositories, while the popular press was distracted with speculations that telomerase could attenuate organismal aging and promote longevity in humans. A more guarded view (12) raised concerns that unscheduled telomerase expression in vivo may lead to an increase in cancer incidence by eliminating replicative senescence, hence obviating a potential tumor suppression mechanism.
The simple interpretation that hTERT expression alone can endow all cell types with unlimited growth potential has given way to a more complex story since the finding that immortalization of mammary epithelial cells and keratinocytes required not only hTERT expression, but also compromise in the RB pathway (13). Moreover, the observation that activated RAS and RAF signals can induce cellular senescence in pre-senescent primary fibroblast cultures well before telomeres have reached a critical length suggests that some physiological stimuli may be capable of acting dominantly to subvert the actions of hTERT (14, 15). Together, these findings raised concerns as to whether the life-extended hTERT-expressing fibroblasts and RPEs also had sustained additional genetic lesions.
Two recent reports have gone a long way in addressing these important issues (10, 11). An extensive molecular and biological characterization of hTERT-immortalized fibroblasts and RPEs now indicates that they behave like their normal pre-senescent counterparts, harboring an intact RB pathway, functional DNA damage checkpoints, and normal karyotypes while lacking well-established hallmarks of neoplasia such as reduced serum requirements, anchorage-independent growth, and tumor formation in nude mice. The non-oncogenic nature of hTERT is in accord with the inability of this gene to substitute for the immortalizing oncoprotein Myc in the classical Myc/RAS cotransformation assay (16).
Why, then, are fibroblasts and RPEs different from mammary epithelial cells and keratinocytes? Part of the answer may lie in the simple fact that amounts of [p16.sup.INK4a] [a critical inhibitor of the RB pathway and key mortality gene (17, 18)] are low in fibroblasts, thus perhaps making it easier for telomerase alone to bypass senescence in those cells. A more likely explanation, however, could relate to cell type-specific differences in the signaling responses activated upon adaptation to culture and how those responses ultimately affect mortality pathways, particularly those governed by [p16.sup.INK4a] and its surrogate pRB. These cell culture-based studies underscore the need to frame these questions in a more physiological context, in which the long-term consequences of broad somatic TERT transgenic expression can be monitored.
It is nevertheless reassuring to know that the hTERT gene is not behaving as a conventional oncogene and that its effects are restricted to telomere metabolism. But do these findings exonerate telomerase as a culprit in cancer? Should we view it now as a “good” enzyme that can be used ad libitum to manipulate the life-span of human cells? Can we look forward to the repair of human tissues and rejuvenation of stem cell populations based on telomerase therapy? If telomerase does not conspire in the tortuous pathway of human tumorigenesis, why then is the enzyme activated in so many cancers? Although telomerase activity is associated with high proliferative rates in some cell types, such regulation fails to explain the appearance of hTERT in most cancers (19). After all, many normal human cell types do not express telomerase while they proliferate in vitro, but tumors derived from such cells do.
Could telomerase simply be a harmless by-product of one of the oncogenes causing malignant transformation? In this regard, it has recently emerged that the transcription of the hTERT gene is regulated directly by the immortalizing oncoprotein Myc (16, 20), whose up-regulation is an obligate feature of virtually all human cancers. Is telomerase just an innocent passenger driven by c-Myc but not lending any growth advantage to tumor cells? This view is made unlikely by the finding that inhibition of telomerase or experimental interference with telomere function arrests and often kills cells even if they are transformed (21-23). Thus, telomerase activity would appear to make an important contribution to the viability of transformed cells, but its action does not fit the usual roles ascribed to oncogenes and tumor suppressors.
Instead of gas pedal or brake, telomerase and more specifically telomeres may be best viewed as the gasoline tank. Gasoline is not sufficient to drive or accelerate the car, nor does it affect the brakes, but when the gas is used up the car stops regardless of the status of its brakes or how hard one steps on the gas pedal. At times, other braking systems may operate early; such is the case with RAS-induced activation of the p16/Rb pathway or other oncogenic signals (14, 15). In those cases, telomerase introduction alone does not immortalize the cells, because they also must overcome the cell cycle arrest. That is, telomerase is not sufficient for transformation, but cells will have indefinite replicative capacity upon telomerase activation if there is a drive for proliferation and if nothing else arrests the cells.
The major remaining challenges are to determine whether shortening of somatic telomeres really constitutes a tumor suppressor mechanism in vivo and to assess the actual contribution of telomerase to cancer. Answers to these questions could emerge from several mouse models: the telomerase knockout mice (24, 25) and mice transgenic for telomerase in experimental settings where telomeres are limiting, in which it can be determined whether there is increased incidence of spontaneous or carcinogen-induced cancer. Ultimately, the definitive answer may have to come from the use of telomerase inhibitors in cancer patients. Although a complete understanding of the role of telomeres in cancer seems far off, the continued interest in telomerase should provide sufficient fuel to carry us to the end of this journey.
References and Notes
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(4.) A.G. Bodnar et al., Science 279, 349 (1998).
(5.) H. Vaziri and S. Benchimol, Curr. Biol. 8, 279 (1998).
(6.) E. Hara, H. Tsurui, A. Shinozaki, S. Nakada, K. Oda, Biochem. Biophys.
Res. Commun. 179, 528 (1991).
(7.) J. Shay, O. Pereira-Smith, W. Wright, Exp. Cell. Res. 196, 33 (1991).
(8.) H. Land, L. Parada, R. Weinberg, Nature 304, 596 (1983).
(9.) C. M. Counter et al, EMBO J. 11, 1921 (1992).
(10.) X.-R. Jiang et al., Nature Genet. 21, 111 (1999).
(11.) C. P. Morales et al., ibid., p. 115.
(12.) T. de Lange, Science 279, 334 (1998).
(13.) T. Kiyono et al., Nature 396, 84 (1998).
(14.) M. Serrano et al., Cell 88, 593 (1997).
(15.) J. Zhu, D. Woods, M. McMahon, J. Bishop, Genes Dev. 12, 2997 (1998).
(16.) R. Greenberg et al., Oncogene 18, 1219 (1999).
(17.) M. Serrano, E. Gomez-Lahoz, R. DePinho, D. Beach, D. Bar-Sagi, Science 267, 249 (1995).
(18.) M. Serrano et al., Cell 85, 27 (1996).
(19.) C.W. Greider, Proc Natl. Acad. Sci. U.S.A. 95, 90 (1998).
(20.) J. Wang, L. Xie, S. Allan, D. Beach, G. Hannon, Genes Dev. 12, 1769 (1998).
(21.) J. Feng et al., Science 269, 1236 (1995).
(22.) B. van Steensel, A. Smogorzewska, T. de Lange, Cell 92, 401 (1998).
(23.) J. Karsleder, D. Broccoli, Y. Dai, S. Hardy, T. de Lange, Science, in press.
(24.) M.A. Blasco et al., Cell 91, 25 (1997).
(25.) H.W. Lee et al., Nature 392, 569 (1998).
(26.) R.A.D. is an American Cancer Society research professor. Work in the
laboratories of T.d.L. and R.A.D. is supported by NIH grants CA76027 and HD
348880, respectively. Article A54710893
Fountain of youth? (telomerase enzyme inhibiting aging)
Abstract: Telomerase is an enzyme capable of preventing aging in cells. These enzymes seem to stabilize the telomere length and maintain the viability of cells. One drawback doubting the efficacy of telomerase in preventing aging is its role in cancer cell proliferation.
A cellular enzyme with rejuvenating powers is providing clues about cancer and how to reverse the aging process.
Normal human cells are mortal. After they divide 50 to 100 times, they get old. Or, as scientists put it, they senesce. Senescent cells are bigger than young cells, excrete proteins at a different rate, and no longer divide.
About a year ago, a team of biomedical researchers announced that they had discovered a way to prevent cells from aging. Jerry Shay and Woodring Wright, professors of cell biology and neuroscience at the University of Texas Southwestern Medical Center in Dallas, took cells from foreskins (byproducts of circumcisions) and added a gene that causes cells to produce an enzyme called telomerase.
Normally, foreskin cells divide about 60 times before becoming senescent. But in this case, the cells have already divided more than 300 times and show no sign of stopping at all.
Nor do they show any sign of abnormally. “With telomerase, cells are like the Energizer bunny,” says Shay. “They just keep going and going and going.”
Meanwhile, at the Geron Corporation in Menlo Park, California, researchers collaborating with Shay and Wright have done similar experiments with cells from human retinas. These cells also appear to have become immortal.
Researchers, though hopeful, don’t yet know whether this method for putting cellular aging on hold will eventually be useful in slowing the aging of the human body, so nobody is suggesting that we all start adding telomerase to our Corn Flakes. In fact, telomerase has a darker side: It is found in 85 percent of all cancers, and it may be the reason cancer cells proliferate out of control. But if researchers can gain a better understanding of how telomerase works, they may be able to develop methods for thwarting both aging and cancer.
In 1961, a cancer researcher named Leonard Hayflick discovered that normal human cells are mortal. He also noticed that the oldest cell cultures in his lab usually died first. Hayflick suspected that cells contain some sort of clock that tells them when it is time to stop dividing. Later he discovered that cells have what he calls an “event counter,” which measures the number of cell divisions rather than the passing of time. If a cell culture is frozen for decades and then thawed, it will resume its doubling at exactly the point where it left off.
Scientists now think they know where the counter is - on the ends of the chromosomes. The chromosome tips are called telomeres, and they consist of thousands of identical sections of DNA strung together like beads on a necklace. Each time the cell divides, some of the beads are lost. And finally, when the telomeres reach a certain length, the cell stops dividing.
In the early 1970s, a Russian immunologist named Alexey Olovnikov proposed a theory for why cell division ceases. Olovnikov was waiting for the subway in a Moscow station when he had a revelation: Imagining that the train track was DNA, and that the engine was the mechanism for replicating the DNA, he observed that the engine would never make a complete pass over the beginning and end sections of the track. For the track to be replicated in its entirety, it would need a “buffer” at each end. Olovnikov theorized that telomeres perform this buffering function.
Evidence that the length of our telomeres has something to do with how quickly we age comes from studies of people with progeria, a mysterious syndrome that causes premature aging. The average life span of progeria patients is 12.7 years. These “children without childhoods,” who look like wizened old men and women, are born with short telomeres.
In the laboratory, the enzyme telomerase appears to stabilize telomere length and keep cells youthful. But in the human body, it may not be that simple. A lot of changes occur in cells as they age, and most scientists doubt that one enzyme can reverse all those changes. But the fact that telomerase can make some cells immortal in the lab has stirred up a lot of excitement.
It has also stirred up some fears. When he first heard the news, Robert Weinberg of MIT’s Whitehead Institute for Biomedical Research reportedly had this to say: “Great! Nice way of making cancer!” Weinberg fears that telomerase could breathe new life into pre-malignant cells that would otherwise be too old to cause trouble. “I think it could unleash a whole torrent of malignant changes,” he warns.
And that is the paradox of telomerase: While it may have the power to rejuvenate old or damaged cells, it may also promote the uncontrolled growth of cancer cells. Indeed, telomerase is found in most, though not all, cancer tumors. But therein may lie a potential weapon against cancer:
By neutralizing the activity of telomerase, researchers hope to “re-mortalize” cancer cells and make them easier to kill.
Geron is already working to develop drugs that would inhibit telomerase. Nobody has yet tried a telomerase inhibitor in human patients, however. “It would clearly be unrealistic to expect that a telomerase inhibitor will be a magic bullet,” says Geron chief scientist Calvin Harley.
As Harley and his collaborators see it, telomerase itself probably doesn’t cause cancer. High levels are found in reproductive tissues such as testicles, but testicular cancer rates aren’t unusually high. More likely, telomerase makes it possible for cells that are already cancerous to continue dividing.
Even if a telomerase inhibitor can’t cure cancer, monitoring the substance may make early detection easier. High levels of telomerase found in urine, blood, or Pap smears could be an indicator of cancer. It might be possible to detect bladder cancer, for example, using a urine test instead of an invasive probe. Telomerase monitoring might also help detect recurrences of cancer after treatment.
In any case, telomerase is teaching scientists much about the relationship between cancer and aging. Many experts in the field now believe that cell senescence may have evolved to keep us tumor-free during our reproductive years. Limiting the number of times a cell can divide also limits the number of mutations that can occur. But the average American now lives well past reproductive age, and this defense doesn’t protect us in later years.
In fact, different strategies may be appropriate for different stages of life. One approach might be to take telomerase activators while we are young, and then switch to telomerase inhibitors to prevent cancer later in life. But it will be many years before such choices are available.
“The telomerase era is now less than a year old in terms of real applications,” explained Weinberg at a recent telomerase conference. “This is just the beginning.”
At the tips of our cells’ chromosomes are structures called telomeres, “caps” that protect the tips from damage. These caps deteriorate as a cell ages, and eventually the cell can no longer divide. The enzyme telomerase confers immortality on some cells, such as cancer cells, by maintaining the caps.
Stem cells, present in the earliest stages of developing embryos, are the parent cells of all tissues in the human body. Telomerase maintains the telomeres of these cells at a stable length, giving the cells the ability to reproduce continuously.
NORMAL SOMATIC CELL
In most of the body’s differentiated cells, telomeres is not present, and telomeres are no maintained at a prescribed length. Each time the cell divides, and its chromosomes are replicated, the end segments of the telomeres are lost. As a cell gets older, its telomeres become shorter and shorter.
Telomerase is found in most cancer cells. It maintains telomere length so that cancer cells can continue dividing endlessly. Telomerase inhibitors may “re-mortalize” cancer calls by allowing their telomeres to deteriorate.
After a somatic cell has divided 50 to 100 times, its telomeres become critically short. Without telomerase to restore the telomeres, the cell becomes senescent. Such “old” cells are not dead, but they can no longer divide.
Mag.Coll.: 97D0560 Article A53650101
Science, Jan 8, 1999 v283 i5399 p154(1)
(telomerase gene raises hope in cancer research)
In ancient Greece, immortality was the province of the gods, who spun the length of each lifetime. But last year it was scientists who rendered normal human cells immortal, by adding the gene for a chromosome-capping enzyme called telomerase (Science, 16 January 1998, p. 349). The achievement raised hopes that the telomerase-immortalized cells might be used to replace cells lost to injury or diseases such as diabetes and rheumatoid arthritis. But that promise was tempered by a big concern:
Because telomerase prevents normal cell senescence—one of the cell’s several safeguards against cancer—the altered cells might turn cancerous once in the body.
Now, the same researchers who created the cells show that they can grow—perhaps forever, at least in lab cultures—without displaying the typical signs of cancer. Some researchers caution, however, that the new work hasn’t removed all the worries about using the cells in therapy.
The researchers doing the work, including Jerry Shay and Woodring Wright of the University of Texas Southwestern Medical Center in Dallas and Choy-Pik Chiu of Geron Corp. in Menlo Park, California, turned to telomerase to try to overcome a natural barrier: Normal cells divide only a limited number of times in culture. That meant that efforts to replace tissue lost to injury, disease, or aging by removing healthy tissue, growing it in the laboratory, and transplanting it back into the body are often impractical. Researchers had traced the difficulty to the shortening of the cells’ telomeres, specialized DNA structures that stabilize the ends of chromosomes. The telomeres ebb away with each cell division until the cells become senescent and eventually die.
Telomerase, which can rebuild telomeres, is not made by most normal cells. But about a year ago, Shay, Wright, Chiu, and their colleagues found that adding an overactive version of the telomerase gene to foreskin fibroblasts and retinal epithelial cells extended their life-spans by more than 25%. The cells are still going strong after three times their normal lifetimes, Shay says.
To allay fears that transplanting such immortalized cells into the body might open a Pandora’s box of cancer, the Texas and Geron groups, now working independently, tested the cells for other telltale traits of cancer cells.
These include the ability to continue growing when their DNA is damaged, when they are in contact with other cells, or when deprived of calf serum and the growth factors it contains—all conditions that stop normal cells in their tracks. The two groups found none of these abnormalities in the telomerase-immortalized cells, nor did they see any of the chromosomal changes, such as loss of whole or partial chromosomes, that are characteristic of cancer cells.
The cells also failed to form tumorlike colonies, as cancer cells do, when suspended in a jellylike medium called soft agar, even after two key growth-suppressing genes, p53 and pRB, were inactivated. And they did not form tumors—or grow at all, for that matter—in susceptible mice. Taken together, the two groups’ papers, which appear in the January issue of Nature Genetics, show that key checkpoints on cell growth are still intact in these cells, says cancer biologist John Sedivy of Brown University: “I think it’s a very significant piece of work.”
Cancer experts caution, however, that these experiments don’t eliminate the possibility that the cells will become malignant in humans. “We don’t know that and we can’t know that from these experiments” because of the differences between mice and humans, says cancer biologist Robert Weinberg of the Massachusetts Institute of Technology. Indeed, cancer biologist Al Klingelhutz of the Fred Hutchinson Cancer Research Center in Seattle points out that while Geron and other companies are pursuing telomerase blockers as potential treatments for tumors, “these same researchers contend that immortalized cells are still normal and could be used for treatment of age-related disease. Is it really possible to have your cake and eat it too?” he asks.
Shay and Calvin Harley, chief scientific officer of Geron, respond that it may very well be. To make sure that telomerase-containing cells aren’t malignant, they are doing further tests, such as seeing how many additional mutations it takes to make the cells cancerous. And as a further safeguard, Harley says, Geron plans to put telomerase on a tight leash in replacement cells for damaged tissue: Rather than using a perpetually active telomerase, the company plans to add regulatory sequences to the gene that would enable it to be turned on and off at will by drugs.
Another obstacle besides possible malignancy may limit the use of the technique, however: Telomerase may not immortalize all cell types, Weinberg and other experts say. But Harley says preliminary results suggest that the enzyme can do the job once researchers figure out how to grow the cells properly in culture.
Clearly, much more work will be needed to find out whether telomerase-expressing cells will prove useful in the clinic. But if they do, then using them to overcome tissue damage would result in more than a Pyrrhic victory.
Dan Ferber is a writer in Urbana, Illinois. Article A54396797
Abstract: Research at Geron Corp shows that normal somatic cells can be made immortal by extending the length of their chromosome tips with the enzyme telomerase. This finding confirms the notion that telomeres function as cellular clocks that age as they shorten.
Elegant experiments confirm long-held theory of cellular aging. They have found a way to reverse the aging process,” Tom Brokaw proclaimed on the NBC Nightly News on January 14, 1998. Brokaw was referring to experimental results from researchers at Geron Corporation, in Menlo Park, California, and at the University of Texas Southwestern Medical Center at Dallas. The work showed that providing normal somatic cells with the enzyme telomerase extends the length of their chromosome tips (telomeres) and renders them immortal, yet healthy.
Although the reported results were not quite as dramatic as the fountain of youth that Brokaw evoked, they did confirm the long-held theory that telomeres function as a cell division clock, ticking down time as they shorten. And as years of discoveries coalesce into the burgeoning field of telomere biology, potential medical applications are already on the horizon in such diverse areas as diagnosing cancer, slowing degenerative diseases of aging, and making organ transplants safer.
Human telomeres are made up of repeats of the DNA sequence TTAGGG. Today it is known that telomeres, long thought to protect chromosomal integrity in some vague way, erode with each cell division, ultimately reaching a threshold length that signals division to cease. For many somatic cell types, an end to cell division means not death, but rather the onset of a defined differentiated, or specialized, state. Telomere shortening seems to be the normal default option for most somatic cells. By contrast, cells in the germline and in highly proliferative tissues, such as bone marrow and the epithelium of the small intestine, continually replenish their chromosome tips. So do most cancer cells, which are notorious for having long telomeres and active telomerase.
Chromosomes keep from shrinking in some cells, including cancer cells, thanks to telomerase, a ribonucleoprotein with three components: a catalytic protein portion with reverse transcriptase activity, an RNA template, and an associated protein called telomeric repeat binding factor. The RNA template contains the sequence CCCUAA, the reverse complement of the TTAGGG DNA that forms the telomere. Reverse transcriptase allows the cell to make DNA copies of this RNA template. And the telomeric repeat binding factor brings the chromosome tip, the RNA template, and the reverse transcriptase physically together, so that six-nucleotide DNA repeats can be added to the chromosome end. This cellular machinery is essentially a built-in telomere factory that is turned on only at certain times and in certain cells.
Thoughts on telomeres
The 1980s and 1990s have seen the elaboration and dovetailing of the molecular details of the telomere story. But the saga began many years earlier, with several seemingly unrelated observations made by some of the heavyweights in the history of biology.
Cytogeneticists first noted the importance of telomeres in the first half of the century, when they observed that chromosomes that lost their tips stuck together and vanished after mitosis. H. J. Muller observed the protective effect of telomeres in Drosophila in 1938, and Barbara McClintock did so a year later in corn. In 1961, Leonard Hayflick contributed information that would prove to be pivotal in telomere biology:
He found that cells in culture divide a finite number of times, usually 40-60. This number became known as the “Hayflick limit.”
A decade after the discovery of the Hayflick limit, Alexey Olovnikov, a senior researcher at the Institute of Biochemical Physics and the Russian Academy of Sciences in Moscow, proposed that telomere shrinkage is a countdown to cellular senescence. In 1973 he published “A theory of marginotomy: The incomplete copying of template margin in enzymatic synthesis of polynucleotides and biological significance of the phenomenon,” in the Journal of Theoretical Biology. What that mouthful means is that chromosome tips whittle down because DNA polymerase cannot copy the very end of one of the replicating strands, the so-called lagging strand. Consequently, the chromosomes would shorten at each round of replication. Olovnikov wrote: “Marginotomy causes the appearance, in the daughters of dividing cells, of more and more shortened end-genes.... After the exhaustion of telogenes the cells become aged.” Olovnikov explains that, as he proposed in his 1973 paper, “The telomere shortening could serve as a counting mechanism, which, like a molecular bookkeeper, counts the number of cell doublings already performed.” A bacterium avoids the problem of shrinking chromosome tips, he also noted at the time, because its chromosome is a circle.
Soon after Olovnikov’s prescient hypotheses were published, James Watson published similar ideas. However, Watson’s description of the “end-replication problem” referred to the replication of the ends of linear bacteriophage DNA, and not to aging. Nevertheless, “the telomere field has always cited Watson, since his was the prediction of chromosome shortening that people in the United States knew about, and were testing,” relates Carol Greider, of the Johns Hopkins University School of Medicine. “I did not know of Olovnikov when I discovered telomerase, nor did others,” Greider says, although she adds that researchers in the field of aging were aware of his work.
But Calvin Harley, now vice president for research at Geron, knew of Olovnikov’s work and spread the word by describing and referencing it in a 1991 publication in Mutation Research. “Experiments have validated his predictions,” Harley says. “But Olovnikov didn’t know the biochemistry of DNA replication very well and couldn’t describe it. He was thinking about aging. Conversely, James Watson carefully defined the end-replication problem, and wrote nothing about aging.” Many telomere biologists now concur that both of these researchers deserve credit for identifying the phenomenon of telomere shrinkage.
Experiments reveal how telomeres shrink.
Olovnikov suggested that an enzyme might maintain chromosome ends. “But molecular tools were needed to prove this idea,” Harley says. A living system in which to explore telomere behavior came in the form of the ciliated protozoan Tetrahymena thermophila. This pond organism provides an enriched system for probing telomeres because when it forms sex cells, its chromosomes fragment and then replicate, generating about 20,000 telomeres. (By contrast, a human somatic cell has just 92 telomeres.) In 1978, Joseph Gall and Elizabeth Blackburn, then at Yale University, found that telomeres in T. thermophila consist of many short DNA repeats. By the mid-1980s, Blackburn, at the University of California-Berkeley, and Greider, then a graduate student in Blackburn’s lab, had discovered and described the enzyme that extends telomeres, naming it telomerase.
In 1986, Howard Cooke, at the Medical Research Council in Cambridge, UK, measured telomere length in human chromosomes, noting that the tips were shorter in somatic cells than in sperm cells. By 1989, Robert Moyzis and coworkers at Los Alamos National Laboratory identified the human telomere repeat as TTAGGG, and in 1990, Harley, Greider, and Bruce Futcher found that the telomeres of human somatic cells shorten as the number of cell divisions increases, although those of cancer cells do not.
In the early 1990s, several observations solidified the link between telomere shortening and aging. In 1992, Richard Allsopp and colleagues at Geron reported in Proceedings of the National Academy of Sciences (89:10114-10118) that children with the rapid-aging disorder Hutchinson-Gilford syndrome have unusually short telomeres. In 1993, shorter-than-normal telomeres were found in people with Down’s syndrome, and 3 years later, they were also found in people with Werner syndrome, an adult-onset rapid-aging disorder. Cells from people with these disorders literally race through their allotted divisions, accelerating life at the cellular level as the body ages ahead of schedule. (People with Down’s syndrome have a shortened life span and often develop early-onset Alzheimer’s disease.)
In 1995, Geron researchers and Greider’s group at Cold Spring Harbor reported in Science (269: 1236-1241) that they had cloned the RNA template component of human telomerase - an 11-nucleotide sequence that includes the critical CCCUAA that encodes the telomere repeat. They and others subsequently measured the amount of RNA template in various tissues, finding greater abundance in tumor cell lines and germline tissues than in somatic cells.
Researchers also probed the origins of the telomerase system by searching for clues in reverse transcriptase, an enzyme that is not unique to telomerase. RNA viruses use reverse transcriptase to copy themselves into DNA in host cells, and retrotransposons also use the enzyme. A retrotransposon is a piece of moveable DNA that transcribes itself into an RNA intermediate when it changes location, and then reverse transcribes itself back into DNA when it inserts at a new location in a chromosome.
The fruit fly has retrotransposons rather than typical telomeres at its chromosome tips. “If a retrotransposable element wanted to use as a target the end of a chromosome, it would effectively take over the role of telomerase. This is what has apparently occurred in Drosophila melanogaster,” says Thomas Eickbush, of the University of Rochester, in New York. He discussed the evolutionary relationship between retrotransposons and telomerase - that is, which came first - in 1997 in Science (277:911-913). Eickbush suggested that, in early eukaryotes, telomeres originated from retrotransposons, which a retrovirus perhaps supplied, and that the unusual telomeres of Drosophila reflect a more recent takeover of somatic cells by retrotransposons that preferentially insert at chromosome ends.
Revealing the role of telomerase
Two key experiments reported in late 1997 and early 1998 further strengthened the connection between telomere shortening and cell senescence, while indicating that the enzyme’s role in cancer causation is complex. One investigation removed telomerase in knockout mice and observed the onset of senescence. The other work, which made the nightly news, added telomerase to human cells in culture and demonstrated extension of the cells’ proliferative lifetime.
Mice in which telomerase was eliminated by deleting (or “knocking out”) the telomerase RNA component gave Greider and her colleagues at Cold Spring Harbor, the Albert Einstein College of Medicine in New York City, and Quest Diagnostics, Inc., in Teterboro, New Jersey, the opportunity to ask what life would be like without telomerase. The researchers created these knockout mice, then observed them and analyzed several highly proliferative tissues through six generations (Cell 91:25-29 and Nature 392: 569-574).
As the researchers had expected, the knockout mice did not fare well. Overall, the lack of telomerase compromised chromosome stability and the integrity of cells that normally divide often. Their telomeres became shorter than normal, their chromosomes broke, and some nonhomologous chromosomes fused to form translocations. The animals’ fertility plummeted, reproductive organs shrank, and highly proliferative tissues, such as testis, spleen, and bone marrow, degenerated. These results therefore confirmed that telomerase is important for maintaining highly renewable tissues. Interestingly, cells cultured from the knockout mice could still become cancerous. This result showed that telomerase by itself does not cause cancer, an observation that is consistent with the fact that cancer development is often a multistep process requiring the participation of several genes.
In the 16 January 1998 issue of Science, Harley and colleagues at Geron, and Woodring Wright, Jerry Shay, and colleagues at Southwestern Medical Center, reported the effects of adding the gene that encodes human telomerase reverse transcriptase to normal human cells in culture. These experiments used cells important in human disease and aging-retinal pigment epithelium, fibroblasts, and vascular endothelium. Slowed metabolism of retinal epithelium can cause age-related macular degeneration. Fibroblasts in aging skin make less collagen and elastin and more collagenase, causing wrinkles. And overgrowth of the endothelium that forms capillaries and lines blood vessel interiors contributes to atherosclerosis.
The results of adding telomerase to these cells were striking - the cells regained their proliferative potential, ignoring the Hayflick limits. “For the first time, we showed that if you highly specifically modulate telomere dynamics, you can see the predicted effect on cell lifespan. It proves the causal relationship between telomere length and aging,” says Harley.
The fact that most cancer cells have active telomerase and long telomeres led to the hypothesis that telomerase is required for tumor growth, with telomere shortening in normal somatic cells having a tumor-suppressing function. However, although many of the cells to which the researchers added telomerase reverse transcriptase churned out the enzyme at levels similar to those of cancer cells, signs of cancer have not appeared, and the cells seem normal despite ignoring the Hayflick limit. “After a year, the cells have not progressed to cancer. They have normal karyotypes, pass all the cell cycle checkpoints, and do not cause tumors when injected into nude mice [which lack immunity and are used to test tumor-forming potential]. They divide at a reasonable rate, and they have a youthful appearance,” Shay reports.
The fact that the telomerase-bolstered human cells do not become cancerous, and that telomerase-deficient mice can still get cancer, is not as contradictory as it might seem. It just shows that telomerase is neither necessary nor sufficient to cause cancer. “If we’ve learned anything over the past 20 years, it is that a lot of different insults are required to transform a normal cell to a cancerous cell. By simply adding telomerase, you’re only affecting one factor. As long as the other pathways are intact, there is no reason to expect an increase in cancer incidence,” Shay says. Telomerase may enable a cell to ignore the Hayflick limit, or directly or indirectly destabilize chromosomes, which in turn could activate an oncogene or deactivate a tumor suppressor gene that is part of the pathway to cancer. “Now we have to see how telomerase fits into all the other aspects of cancer that are controlled by other genes,” Shay adds.
With the components of telomerase clearly identified, and the enzyme’s function elegantly demonstrated, the next stage in the continuing tale of telomeres will be developing clinical applications.
Because telomerase is critical to maintaining cellular stability and cell division, altering this enzyme’s activity may have varied uses. In basic research using cell cultures, “controlling telomerase would yield a
constant source of human cells that are not cancerous, but would proliferate,” Shay says. In clinical applications, new understanding of telomerase function could lead to more sensitive cancer diagnostics and make transplants safer, treat AIDS, and perhaps even rejuvenate a Measuring telomerase levels, for example, can be used to track cancer progression. In one study, 12 of 16 children with neuroblastoma and high telomerase activity in their cancer cells died, whereas only 2 of 60 children with low telomerase activity died. “About 85 percent of tumors contain this marker, and use of telomerase as a cancer marker is already a routine procedure in some oncological centers,” Olovnikov says. A polymerase chain reaction-based assay called TRAP (telomeric repeat amplification protocol) can spot a single telomerase-producing cancer cell among 10,000 healthy cells, and a technique using fluorescent in situ hybridization (FISH) and flow cytometry, called “flow-FISH,” can measure telomere length. Clinicians may someday manipulate telomerase level or activity as a way to treat cancer, but the exact role of telomerase in cancer needs to be worked out first.
New understanding of telomere biology may also solve a vexing problem with bone marrow transplants: Something about the transplant process seems to rev up the mitotic clock, accelerating the aging of donor cells. Robert Wynn and colleagues at The Paterson Institute for Cancer Research in Manchester, UK, reported in the 17 January 1998 issue of The Lancet that telomeres in transplanted bone marrow cells are shorter than those in normal bone marrow cells in either the donor or the recipient. Rosario Notaro and coworkers at Memorial Sloan-Kettering Cancer Center in New York City reported in the 9 December 1997 issue of PNAS that the more cells that are transplanted, the less the telomeres shrink. It is as if transplanting only a few cells stresses them in their effort to repopulate the recipient’s marrow, and in response the cells age faster than normal, the researchers suggest.
The rapid aging of transplanted tissue may explain the increased risk that bone marrow transplant recipients face of developing blood cancers years later, Shay suggested in an editorial accompanying the Lancet article. “A bone marrow transplant is supposed to be all stem cells, but this is not completely so. Ten to 15 years later, a recipient may develop leukemia because the transplanted cells did not have the proliferative capacity of a true stem cell,” he says. Inserting telomerase into the donor bone marrow cells before the transplant may help to extend the cells’ lifetimes.
A similar approach of extending cellular life with telomerase might be used to treat AIDS, but in this case the patient’s own cells would be used. The human body has enough hematopoietic (blood-forming) stem cells to last a lifetime, but as HIV kills more and more T cells, the stem-cell population has to work overtime to replace them. The hematopoietic system may eventually shut down. “Telomere biology might be part of the AIDS story,” Shay says. Instead of transplanting bone marrow from donors, hematopoietic stem cells could be taken from a person at the early stage of HIV infection. The cells would have their telomeres extended in culture. Then, when the patient’s T-cell count falls as the infection progresses, he or she can receive the stored stem cells, which may replenish the T-cell supply.
Telomere biology may also be exploited to address signs of aging. A blast of telomerase might keep fibroblasts in the skin’s dermis layer at a more youthful stage, in which they synthesize collagen and elastin rather than collagenase. Reactivating collagen and elastin production from within might be an alternative to injecting bovine collagen to plump out wrinkles.
Further in the future is the possibility of using autologous (self) cell implants to renew selected tissues that degenerate with age, approaching Tom Brokaw’s view of telomere biology as providing a fountain of youth. Olovnikov speculates that “such cells will be treated in vitro with telomerase activity-containing viral vectors. Artificially elongating their telomeres will preserve these cells”normalcy,’ so they will not senesce. Such cells might be used to renew the inner parts of blood vessels, cells of the pancreas, or even normal postmitotic cells such as cardiocytes and neurons.” But those sorts of applications are still very much in the future. As Shay sums up, “We can’t make people live forever. There’s nothing wrong with fantasizing, but there are too many interesting short-term benefits of the research to focus on. If we can develop tissue-specific therapies, if we can correct certain problems, then maybe we will live longer.”
Ricki Lewis is the author of several life science textbooks published by McGraw-Hill and is working on a book on scientific discovery. Article A53392373
Castration Induces an Increase in the Expression of Telomerase Activity in the Prostate of Nonhuman Primates
“Castration Induces an Increase in the Expression of Telomerase Activity in the Prostate of Nonhuman Primates.” N. Ravindranath, S.L. Ioffe, G.R. Marshall, S. Ramaswamy, T.M. Plant and M. Dym. Georgetown University Medical Center, Washington, DC; University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania.
According to an abstract submitted by the authors to the 80th Annual Meeting of The Endocrine Society, held June 24-27, 1998, in New Orleans,
Louisiana, “It has recently been demonstrated that telomerase, a ribonucleoprotein that synthesizes the telomeric repeats of TTAGGG de novo and adds them to the ends of mammalian chromosomes, may play a pivotal role in the aging process and in the progression of cancer. Telomerase activity is not detectable in the normal human prostate. However, the majority of human prostate cancer tissues exhibit telomerase activity. Since androgens play a major role in prostate tumorigenesis, it is important to study the
regulation of telomerase activity in the prostate in the absence of testosterone. Using the monkey as a surrogate model for human, we investigated the effect of androgen-depletion on the expression of
telomerase activity in the prostate.
Five normal adult male rhesus monkeys were castrated. Their precastration serum testosterone (T) levels were 3.15 +/- 1.9 ng/ml (mean +/- S.D.). At six weeks postcastration, their T levels fell to 0.20 +/- 0.15 ng/ml (mean +/- S.D.). A control group of five other rhesus monkeys were sham-operated. Their T levels, pre- and post-sham operation, were 2.20 +/- 1.3 ng/ml (mean +/- S.D.) and 2.32 +/- 1.1 ng/ml (mean +/- S.D.), respectively. Six weeks following castration or sham operation, prostate tissues were removed immediately post-mortem. The testes from the sham operated monkeys were also removed at this time. There was no observable change in the size and shape of the prostate glands in the castrated monkeys in comparison to sham operated monkeys.
The anterior, posterior, left-lateral, right-lateral, and median regions of the gland were dissected and frozen immediately. Telomerase activity was assayed by using a polymerase chain reaction-based telomeric repeat amplification protocol. All the five regions of prostate from sham operated animals failed to exhibit telomerase activity. However, the testis expressed telomerase activity. In contrast, extracts from the whole prostate as well as from the five individual regions of the prostate from castrated monkeys expressed very high levels of telomerase activity. In summary, these results indicate that in monkeys androgen ablation leads to upregulation of telomerase activity. The negative regulation of telomerase activity by androgens is probably lost during prostate tumorigenesis. Supported by NIH
Grant HD33728.” Article A20960745
AMA, The Journal of the American Medical Association, June 3, 1998 v279 n21 p1732(4)
Abstract: Two 1998 studies show that scientists may be able to extend life and possibly reduce the frequency of age-related diseases. Formerly, research showed that most cells continue to divide up to a point but ultimately lose this ability and show signs of aging. Cell aging was linked to the shortening of the ends of chromosomes after each division. These ends are called telomeres. The 1998 studies showed that cell aging could be reversed by introducing an enzyme called telomerase into the cells. The enzyme lengthens the telomeres, causing the cell to revert to a younger age.
Recent research has shown that inserting a gene for the protein component of telomerase into senescent human cells reextends their telomeres to lengths typical of young cells, and the cells then display all the other identifiable characteristics of young, healthy cells. This advance not only suggests that telomeres are the central timing mechanism for cellular aging, but also demonstrates that such a mechanism can be reset, extending the replicative life span of such cells and resulting in markers of gene expression typical of “younger” (ie, early passage) cells without the hallmarks of malignant transformation. It is now possible to explore the fundamental cellular mechanisms underlying human aging, clarifying the role played by replicative senescence. By implication, we may soon be able to determine the extent to which the major causes of death and disabililty in aging populations in developed countries—cancer, atherosclerosis,
osteoarthritis, macular degeneration, and Alzheimer dementia—are attributable to such fundamental mechanisms. If they are amenable to prevention or treatment by alteration of cellular senescence, the clinical implications have few historic precedents.
But the lengthening of the thread of life itself, and the postponement for time of that death which gradually steals on by natural dissolution and the decay of age, is a subject which no physician has handled in proportion to its dignity.
Francis Bacon, The Advancement of Learning
THERE IS A natural inclination to believe that scientific and medical advances will be accretional: gradual, predictable additions to our current body of knowledge and practice. Although this is an appropriate view of the overwhelming bulk of day-to-day science and medicine, history also shows unpredicted and innovative transitions in our paradigms and, subsequently, in clinical practice. The most notable of such shifts has been germ theory (with the consequent development of immunizations, antibiotics, and sterile surgical technique). The same is likely to be true of genetics, although, as yet, the clinical benefits remain largely theoretical.
An equally remarkable potential—in regard to cancer, aging, and age-related disease—is highlighted in the startling articles by Bodnar et al and Vaziri and Benchimol, which demonstrate that human cell senescence can be reversed (and the “Hayflick limit” extended) by transfection with a gene for the catalytic component of telomerase. The publication of these results makes it appropriate to review the field and outline the prospects for clinical medicine.
BEYOND THE HAYFLICK LIMIT
In 1961, Hayflick and Moorehead showed that normal somatic cells have a limited replicative potential, roughly 50 in young skin fibroblast cells. Prior to achieving this maximum, they slow their rate of division (to such an extent that they probably rarely reach their replicative limit in vivo) and manifest identifiable and predictable morphologic changes characteristic of “senescent cells.” Since that time, we have defined patterns of predictable changes in genetic expression (“senescence-associated gene expression”) that accompany the replicative block in a variety of cell types.[5-9]
In the early 1970s, Olovnikov and Watson independently pointed out that DNA polymerase primers overlay and “mask” a portion of the terminal chromosome from DNA polymerase; a portion of the telomere is not replicated and therefore shortens with each cell division. During the 1980s, molecular biologists began to address this hypothesis, and by 1990, research showed that telomeres are indeed shorter in senescent cells than in younger cells.[12,13] Since then, results have accumulated[14-16] that (not without exception) imply, but did not prove, that telomere shortening is the clock that results in the shift to a senescent pattern of gene expression and ultimately cell senescence and the Hayflick limit.
This barrier to unlimited cell replication, the Hayflick limit, has been reliably reproduced and has few (and identifiable) exceptions in multicellular organisms. These exceptions include cancer cells,[20-22] the germ cell lineage,[23-27] and certain stem cell lines.
These latter cells (including hematopoietic stem cells, gastrointestinal crypt cells, hair follicles, and perhaps liver cells) also demonstrate replicative senescence, but do so with a much extended cellular life span (a greater number of divisions) compared with other somatic cell types. This extension correlates with transient expression of telomerase[30,31]--a reverse transcriptase comprising (at least) an RNA template and a catalytic protein component--that allows them to slow the rate at which the erosion of telomere bases occurs. Telomerase expression also occurs in the germ cell line and primordial stem cells,[34,35] whose potential clinical applications include the ability to grow tissues and organs without being limited by replicative senescence, as has been the case until now.
The acceptance of cellular senescence raised 2 immediate and critical questions: (1) what is the cellular “clock” that timed cell replicative senescence, and (2) what is the relationship between cell senescence and aging in the organism? Bluntly, does replicative senescence tell us anything clinically useful about the causes of (and therefore potential treatments for) diseases such as cancer, arteriosclerosis, arthritis, and dementia?
After 35 years of research, Bodnar et al have emphatically answered the first of these 2 questions: the telomere is the clock of replicative senescence and it can be reset. The work has been independently confirmed by Vaziri and Benchimol, as well as by others. Telomerase acts by adding DNA bases to telomeres. In addition to its RNA template that is present in cells normally (and which acts as a “die” for the TTAGGG sequence), it contains a catalytic protein component that is not found in normal, aging, somatic cells. In the first published work, Bodnar et al transfected a gene for the catalytic component into such cells and showed that this resulted in extended telomeres. More importantly, however, was that this extended the replicative life span of cells and gave them a pattern of gene expression typical of young cells.
By the time results were submitted, treated cells already showed 40% more population doublings and showed no evidence of slowing their rate of cell division. Although this result was strongly implied by initial work on cell hybrids in which telomere lengthening correlated with extended cell life span, it is the article by Bodnar et al that effectively establishes that telomeres are the clock of replicative aging. Telomeres not only shorten with cell aging, but relengthening the telomere appears to reset gene expression (as measured by expression of [Beta]-galactosidase, an established biomarker of aging in these cells), cell morphology, and the replicative life span. Furthermore, and despite the appropriate concern that this should raise, there is no evidence yet that telomerase expression per se causes malignant transformation.
Remarkable as these implications are, it is far more important in allowing us to begin answering the second question, viz, what does replicative senescence have to do with cancer and other age-related diseases and therefore with their treatment? It is here that the historical transition may be in the offing. The potential is therapeutic modification of the cellular mechanisms underlying age-related diseases to an as-yet-unparalleled and effective degree. These possibilities include effective cancer therapy and potentially, but more distantly, the effective prevention and treatment of atherosclerosis, osteoarthritis, immune senescene dermatologic aging, macular degeneration, and Alzheimer dementia at a fundamental cellular level. While such possibilities have been discussed previously and speculatively, we now have the tools to begin testing them.
IMPLICATIONS FOR UNDERSTANDING DISEASE
The potential impact of altering aging and age-related diseases at the genetic and cellular level is enormous, both medically and economically. Although some hormonal therapies, such as estrogen replacement,[41,42] perhaps growth hormone, and possibly dehydroepiandrosterone (DHEA),[44-46] have evidence of efficacy, their clinical role is as yet poorly defined and their indications are limited. Estrogen replacement, for example, lowers the atherosclerotic death rate in women (and may delay or ameliorate Alzheimer dementia), but is not appropriate in men, may increase the risk of breast cancer[48,49] or endometrial cancer,[50,51] and does not prevent aging overall. With even less to offer and more caveats, human growth hormone, DHEA, and the like may be found to play a clinical role in some aging-related diseases or in the quality of life, but there is no evidence that these hormones (or any others) underlie fundamental aging processes.
Nor should we expect hormones to play such a role. While it is true that hormones show age-related changes, such models implicitly assume that “wear and tear” of the secreting organ assumes the role of “clock.” The more appropriate and intriguing model is that suggested by the correlation between replicative senescence and life span between and within species (as well as in progeric syndromes such as Hutchinson-Gilford disease and Werner syndrome).
With regard to cell senescence, there is a growing literature supporting a central role for cell aging in organismal aging. The “limited model”—that telomeres play a pivotal role in replicative senescence—has been addressed in recent reviews[7,58] and receives significant support in the results of Bodnar et al and Vaziri and Benchimol. The “general model”—that cell aging underlies the general process of aging in the organism—however, remains more speculative. This more general model relies not only on changes in dividing cells (which approach their replicative limits), but on nondividing cells (which depend on cells that do divide) and whose dysfunction ultimately results in clinical disease.
The limited model is relatively simple, although much is still unknown about the mechanisms involved. As cells divide, telomeres shorten, resulting both in senescence gene expression[38,59,60] and in inhibition of cell replication via the cell cycle braking system. Telomere loss is detected as genetic damage and invokes the p53 cascade the failure of which is implicated in oncogenesis. The linkage for the first arm of this mechanism—senescence-associated gene expression—is under debate, but current data suggest that it results from changes in the heterochromatin, conformational changes in the chromosome itself, directly through a checkpoint arrest, or from an interaction of these mechanisms.
In cells that do not show replicative senescence—cancer cells, germ line cells, and some stem cells—telomerase maintains telomere lengths, and, according to this model, thereby prevents cell senescence. Both Bodnar and her colleagues (including Vaziri and Benchimol) have provided substantial proof for this model by showing 2 critical results. Lengthening telomeres results in cells that have longer replicative limits and are “younger” (not senescent) as assessed by their patterns of gene expression, physiologic markers, and morphology. Perhaps more importantly, considering the permissive role (necessary but not sufficient) that telomerase expression appears to play in malignancy, telomerase expression and telomere lengthening per se give no evidence—so far—of altering normal cell cycle control, chromosome complement, or cell morphology, or of resulting in any other markers of malignant transformation.
Furthermore, and as a result of their work, the general model now becomes testable. This model goes further, suggesting that replicative senescence itself times and underlies organismal senescence. Replicative senescence is not solely responsible for clinical aging: there are too many data that environmental factors (eg, tobacco use, oxidative damage, ultraviolet exposure) and genetics (eg, diabetes, hypertension, hypercholesterolemia have major roles in many age-related diseases (such as atherosclerosis). Nevertheless, cell aging probably plays a central role in the timing and course of such diseases through a number of mechanisms. This senescence-associated gene expression model of aging does not suggest that older tissues have a preponderance of (or even necessarily any) cells that have reached their replicative limits; rather it suggests that a sufficient number of senescing cells have an altered pattern of gene expression and that this alteration in cell function is both directly and indirectly responsible for the cascade of processes (such as atherogenesis) that we encounter clinically as “aging” in organs and tissues.
Perhaps the best example (in terms of currently available supporting data) of this is dermatologic aging. Many of the changes that occur in the dermis with age—loss of collagen, increased collagenase, and decreased elastin--are paralleled by the changes in senescent dermal fibroblasts in vitro. Although we lack a comprehensive understanding of how skin aging occurs, the model that senescing cells may contribute to this process is now potentially testable, perhaps by directly modulating telomere length. We might now, for example, attempt to delay senescence in vivo (in dermal fibroblasts) by using telomerase analogs, transfection, or activation of gene expression.
Skin aging is not merely cosmetic, having measurable clinical morbidity, but there is greater clinical interest in the effects of aging in other tissues (and cell types), for example, joints (eg, chondrocytes), the central nervous system (eg, glia and secondarily the nondividing neurons that depend on them), and the immune system (eg, lymphocytes). Current technical prowess is not up to these goals yet, but already allows us to extend telomeres in vitro, which may suffice to permit clonal expansion of human cells for vaccine production or to grow skin cells for burn patients.
Similarly we can now test the ex vivo clinical potential in human immunodeficiency virus disease and marrow transplants, in which replicative senescence may be reset using currently available transfection techniques. Recent work by Shay, Moore, and others, for example, suggests that telomere shortening may play a role in limiting the clonal expansion of bone marrow for transplants. This is precisely the sort of clinical possibility that current technology is most capable of testing, with the potential benefit of more successful bone marrow transplants and better patient survival.
Aging vessels—specifically atherosclerosis and its clinical outcomes (largely myocardial damage and stroke)--are a major cause of death and morbidity in developed countries. Here too, the senescence-associated gene expression model offers to clarify and extend our basic model of atherogenesis and suggests an alternative therapeutic approach. Cellular senescence has been observed in arterial endothelial cells, which are themselves implicated in triggering atheroclerosis. In those portions of the vascular tree under high stress (shear stress due to hypertension or vessel bifurcation, for example), cells show shorter telomeres, attributed in this model to greater cell division and cell replacement in these areas.
Such older endothelial cells have an altered pattern of trophic factor production, part of the cascade of atherogenesis. Dividing cells (vascular endothelial cells) can cause age-related pathology in nondividing cells (myocardial cells): many cells do not divide, yet—in this general model—are critically dependent on cells that do. Once again, the importance of the new research is not that it proves or extends this model of vascular aging, but rather that it opens the door to testing the model if we can extend telomeres in vivo in vascular endothelial cells by transfection or gene activation. Such techniques (in vivo) still lie beyond our grasp, yet are tantalizingly close and possess provocative implications: might we prevent (or to an extent even reverse) atherosclerotic lesions and thus favorably alter their clinical outcomes?
The other major cause of death in developed nations is cancer. Malignant transformation requires that cancer cells evade replicative senescence, making aging and cancer “the double-edged sword of replicative senescence.” This raises the concern that in lengthening telomeres to reverse replicative senescence we might increase the risk of cancer Such a concern arises not because telomerase expression causes cancer, but rather because it allows transformed cells to continue dividing and therefore to attain clinical significance. As predicted by this model, telomerase expression is a superb marker of malignancy,[22,29] but is not itself a cause of cancer, a conclusion that begs testing, as in Bodnar et al and studies like it. In fact, the absence of telomerase in most normal somatic cells may play a protective role, forcing the cancer cells in many would-be tumors to stop dividing prior to becoming clinically significant.
Ironically then, our growing knowledge of replicative senescence also implies a novel potential therapy forcing cancer cells to senesce by inhibition of telomerase activity. Here again, Bodnar et al provide a first step, showing that—so far and as predicted—telomerase activity per se does not show any evidence of causing malignant transformation in this study. This initial study is supported and confirmed by a more extensive recent study, which again finds no evidence of malignancy: telomerase-transformed cells appear to be otherwise normal cells. Equally, these results show that we are growing more adept at using the tools of telomere biology and are thereby moving slowly closer to trials of telomerase inhibitors to treat cancer. Such inhibitors would be used to reinstitute a replicative limit in cancer cells, with the clinical aim of braking further tumor growth and forcing such cells to senesce.
IMPLICATIONS FOR AGING
The possibility of directly undercutting the cellular mechanisms causing age-related diseases by altering the mechanisms of cellular senescence suggests a more distant and unprecedented prospect: we might extend not only the mean life span (as with every clinical advance), but potentially the maximum life span as well. The correlation between species life span and the replicative life-span of cells from such species raises this (perhaps soon testable) possibility. Such a potential increase in life span, while unintentional, would have a pervasive impact on our culture.
Any prevention, postponement, or reversal of replicative senescence may carry implications for aging and age-related diseases, and although this work is extraordinary, it is by no means conclusive. Telomere biology and replicative senescence still abound with uncertainties and superficially contradictory data, as does any field at its inception. For example, there are multiple ways of inducing replicative senescence or senescence like is states (oxidative damage, DNA damage, ultraviolet exposure, the ras oncogene, etc) independent of telomere shortening,[77,78] and there are pathways for extending telomeres without telomerase activity.
Additionally, although senescence-associated gene expression and growth arrest serve to define senescence, there is a great deal still to understand, even when we restrict the discussion to a single cell type within a single species. The telomerase knockout mouse (in which cells do not express the gene for telomerase and the offspring appear normal for several generations) clearly suggests that telomere function differs between species, but does not directly contradict the contribution of senescence-associated gene expression to aging,[80-82] nor does it undermine the significance of the work by Bodnar et al or Vaziri and Benchimol.
The importance of this work does not lie merely in our nascent ability to alter cell senescence. Nor are the implications that we may simply be able to test an increasingly complete and elegant theory of aging. Rather the importance, ultimately, lies in its potential to treat human disease, to alleviate patient suffering, and—raising the possibility “in proportion to its dignity”—that we may alter “the thread of life itself.”
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From the Department of Medicine, Michigan State University College of Human Medicine, East Lansing, and the College of Health Science, Grand Valley State University, Allendale, Mich.
Dr Fossel owns stock in Geron Corporation, a biotechnology firm specializing in telomerase research in the fields of aging and cancer.
Reprints: Michael Fossel, MD, PhD, 9464 Conservation Ave, Ada, Ml 49301 (e-mail: Mfossel@aol.com).
Mag.Coll.: 94C4673 Article A20764420
The Lancet, April 18, 1998 v351 n9110 p1186(1)
Abstract: Chromosomal telomeres may play a key role in cellular aging and death, as well as persistence in cancer cells. Telomeres are the ends of chromosomes, which shorten each time a cell divides and may, thereby, control cellular lifespan. Many cancer cells produce the enzyme telomerase, which protects the chromosome ends from shortening. Researchers at Geron Corp. (Menlo Park, CA) have genetically engineered human tissue cells to express telomerase, and the cells appear to be living longer. Application of this technology could promote tissue healing and inhibit cancer growth.
In January this year, Geron Corporation (Menlo Park, CA, USA) boosted midwinter spirits by announcing that cellular immortality may at last be within reach (Science 1998; 279: 349-52). The Geron team had extended the lifespan of normal human foreskin fibroblasts and retinal pigment epithelial cells by genetically engineering the cells to express telomerase, an enzyme that protects chromosomal ends-telomeres-from fraying during cell division. Although most observers doubt that this work will lead to an extension of human lifespan, the finding opens up new therapeutic possibilities for tackling diseases of ageing and cancer.
In normal somatic cells, chromosomes shorten at each cell division because of inherent limitations in the mechanics of DNA replication-about 200 basepairs of DNA are lost from chromosomal ends at each division. This does not immediately cause a problem since each chromosome terminates in a telomere, a highly redundant structure consisting of thousands of copies of a six-basepair DNA sequence. Every time the cell divides, part of this structure is lost. The possibility that fraying telomeres may act as a slow-burning fuse, limiting the lifespan of somatic cells, has intrigued scientists for the past 10 years.
The idea that cell divisions are counted via telomere shortening is supported by the finding that the telomeres of “immortal” germ cells, which express telomerase, do not shorten during cell division. But Geron’s new finding provides what many view as proof of the link between telomere shortening and cell senescence. Normal somatic cells do not express telomerase, and so grow old and die after a set number of cell divisions.
However, the telomerase-expressing somatic cells engineered by Geron and scientists at the University of Texas Southwestern Medical Center (Dallas, TX, USA) break through the senescence barrier. To date, the cells, which normally senesce at 50-55 divisions, have divided more than 100 times. In addition, says Geron vice-president Calvin Harley, “the cells are staying youthful”. The telomerase-carrying cells are free of the signs of ageing shown by their senescent dishmates. They do not accumulate the age pigment lipofucin or increase in size, and cell-cell contacts remain good.
Harley believes that at the very least this experiment will help focus the field of ageing research. A multitude of factors has been implicated in ageing, including: oxidative damage, accumulation of junk proteins such as amyloid, and gene mutation or changes in chromatin structure. “These [things] occur but are not a limiting factor-this experiment says what’s not important and what is”, explains Harley.
A number of exciting therapeutic applications follow from the ability to increase the lifespan of a person’s own healthy cells: rejuvenated cultured skin cells for treating chronic skin ulceration; retinal pigment epithelial cells for treating macular degeneration; and stem cells for bone-marrow transplantation. The last is important because even stem cells are not truly immortal. Work by Malcolm Moore at the Sloan-Kettering Cancer Center in New York, USA, suggests that bone-marrow cells age the equivalent of 40 years during the process of transplantation.
There are concerns that increasing cell lifespan could trigger cancer. But Harley’s reading of the data is that telomerase plays a permissive rather than an initiating role in cancer. For example, he notes, some cancers that are detected at early stages of development, including neuroblastoma, do not involve immortalisation. Furthermore, it is possible that for therapeutic use, telomerase activity might have to be only temporarily upregulated in order to achieve a transient increase in cell lifespan-the more worrying goal of cellular immortality may not be necessary.
Telomerase may be of only minor importance in the initiation of cancer, but its involvement in later stages is more significant. 85% of tumours express telomerase-a finding that has spurred Geron and other companies to develop telomerase inhibitors. Such drugs, it is hoped, will stop cancer-cell growth without affecting normal cells.
Last year’s cloning of telomerase indicated that the enzyme bore a strong resemblance to retroviral reverse transcriptases, raising hopes that zidovudine-a reverse transcriptase inhibitor used for treating AIDS-could provide a double dividend. But the drug was a poor inhibitor of telomerase, a result that may be explained by a recent analysis showing that the enzyme is more closely related to reverse transcriptases carried by mobile genetic elements (transposons) than to retroviral enzymes.
However, one caveat to the strategy of blocking cancer-cell growth with anti-telomerase drugs comes from work by Roger Reddel and his team at the Children’s Medical Research Institute in Sydney, Australia. Reddel’s work shows that about 5% of tumours have no detectable telomerase activity but nevertheless have long, heterogeneous telomeres.
Reddel believes that these tumours have recruited an alternative mechanism for lengthening telomeres (ALT), a mechanism that might foil attempts to stop cancer-cell growth by using telomerase inhibitors (Nat Med 1997; 3:127 1-74). Such drugs may merely select for cells expressing the ALT mechanism. Indeed, evidence from mouse experiments suggests that the ALT mechanism is all too easily recruited. Last year, teams led by Carol Greider (then at Cold Spring Harbor Laboratory, Long Island, NY, USA) and Ronald DePinho (Albert Einstein College of Medicine, NY, USA) made transgenic mice that lacked the telomerase gene. These mice had chromosomes with short telomeres, but, never theless, cells from these mice could be induced to become cancerous as easily as normal cells (Cell 1997; 91: 25-34).
In the light of these results and his own, Reddel believes that successful cancer therapy will ultimately need drugs targeted against both telomerase and the ALT mechanism- his laboratory is now trying to elucidate the ALT mechanism.
Mag.Coll.: 93K4418 Article A20555719