From: ma157727@student.uq.edu.au (Mitchell Porter) Newsgroups: sci.life-extension Subject: Two telomerase articles Date: 21 Feb 1995 09:39:27 GMT Organization: Prentice Centre, University of Queensland NNTP-Posting-Host: student.uq.edu.au X-Newsreader: NN version 6.5.0 #3 (NOV) Two articles on telomerase from _Science_, reproduced without permission. I have not attempted to reproduce accompanying figures. SCIENCE - VOL. 265 (1656-1658) - 16 SEPTEMBER 1994 CHROMOSOME ENDS CATCH FIRE "The limited lifespan of normal cells and the ability of cancer cells to transcend it may both depend on the telomeres, the structures that cap the chromosome." To a living cell, few jobs are more important than protecting the integrity of its chromosomes. When these libraries of genetic information are damaged, the cell's own survival is in jeopardy, to say nothing of its progeny's. Ever since the work of pioneering geneticists Hermann Muller and Barbara McClintock more than 50 years ago, cell biologists have believed that at least part of the job of chromosomal protection falls to their ends - the so-called telomeres. McClintock's work suggested, for example, that the telomeres keep chromosomes from fusing end-to-end, which could lead to chromosome breakage and loss as cells divide. That might seem a big enough assignment in the life of a cell. But a recent spate of research has indicated that telomeres, now known to consist of short repeated DNA sequences plus associated proteins, play many additional roles that touch both on the normal control of cell proliferation and the abnormal growth of cancer. "From both the scientific and practical standpoint, there's been a huge amount of progress", says Virginia Zakali of the Fred Hutchinson Cancer Research center in Seattle, whose own work focuses on yeast telomeres. The newer roles being assigned to telomeres include aiding in gene regulation and pssibly serving as a "mitotic clock" for the cells of higher animals. Researchers have shown that the telomeres shorten slightly every time the chromosomes replicate in preparation for cell division, suggesting that cells becom senescent and die when the telomeres have shortened beyond a certain point. That shortening takes place because most normal cells do not make telomerase, the special enzyme needed to synthesize telomeres. But in cancer cells, telomerase synthesis somehow becomes reactivated, an event that may contribute to the cells' ability to divide continually. This finding, reported earlier this year by Calvin Harley, Silvia Bacchetti, and their colleagues at McMaster University in Hamiton, Ontario, suggests that telomerase may be a good target for anti-cancer drugs. These findings mark the arrival of telomere research into the broad mainstream of biomedical research. When the field opened up in the 1970s, researchers studied telomeres in protozoan ciliates, single-celled organisms that propel themselves with hairlike projections called cilia, rather than in mammalian cells. At the time, ciliates were much easier to work with because they have many more telomeres per cell than do mammal cells. These organisms have two nuclei, and during the formation of the larger of these, the so-called macronucleus, the chromosomes break up into fragments that then replicate, producing from 20,000 to as many as 10 million pieces of DNA, each of which becomes capped at both ends by telomeres. In contrast, a human cell has but 92 telomeres, two for each of the 46 chromosomes. The ciliates quickly began yielding surprises. Elizabeth Blackburn of the University of California, San Francisco, recalls that when she isolated the first telomere from the ciliate _Tetrahymena thermophila_ in the early 1970s, "I realized [immediately] that there was something quite peculiar about these molecular regions." The work, which was done while she was a postdoc in Joseph Gall's laboratory at Yale University, showed that the _Tetahymena_ telomere consist of a short sequence, TTGGGG (where T and G denote thymine and guanine, two of the four bases whose sequences spell out the genetic information in DNA), repeated some 50 to 70 times. The structure of the telomeric DNA came as a surprise because at the time only DNAs which had been examined closely came from viruses and bacteria, which do not have such repeated sequences. But for telomeres "peculiar" is in fact normal. Researchers soon found that the telomeres of other ciliates also consist of short TG-rich sequences repeating over and over. And as telomere researchers began broadening their view to include other kinds of organisms, they began accumulating evidence that these special structures do indeed help to maintain the chromosomes, just as McClintock's work had suggested. In the early 1980s, for example, Blackburn, working with Jack Szostak at the Dana-Farber Cancer Institute in Boston, showed that DNA strands equipped with _Tetrahymena_ telomeres survive inside yeast cells; ordinarily, yeast cells quickly degrade foreign DNA. And Zakian and her colleagues obtaind similar findings with telomeres from another ciliate, _Oxytricha fallax_. A pleasing resemblance These yeast experiments helped allay one worry: that the telomeric sequences detected on the chromosome fragments might not be at all representative of the telomeres of the more standard chromosomes of other organisms. "The ciliates are a great paradigm," Zakian says, "but there's always the concern that they are abnormal." But the finding that ciliate telomeres protect DNA in yeast, as they apparently do in the ciliates themselves, suggests that telomeres must have been highly conserved during evolution, Blackburn says. Yeast and _Tetrahymena_ are very distantly related; they are not even members of the same kingdom. Confirmation of just how conserved telomeres are came later, in 1988, when Robert Moysis and his colleagues at Los Alamos National Laboratory isolated the first human telomeres and showed that they also consist of a repeating sequence, TTAGGG (where A stands for the base adenine). And since then researchers have found the same repeating sequence at the chromosome tips in every vertebrate they have examined. As the yeast work progressed, it showed that telomeres not only prevent the immediate degradation or loss of the chromosomes, but they also protect chromosomes in a more subtle way. By extending the ends of the chromosomes with repetitive, non-coding DNA, they prevent the gradual loss of genetic information that would otherwise result from a quirk in the way DNA is replicated. The polymerase enzyme that copies the DNA can't reproduce both strands of the double-helical molecule all the way to the ends. As a result, chromosomes would get progressively shorter with every cell division - and essential genes would gradually be eroded. And in the lower organisms, at least, this doesn't happen because the cells can add telomeric DNA to the incompletely replicated ends of the chromosomes. The yeast work showed that when linear DNAs tipped with ciliate telomere sequences are put into yeast cells, they subsequently acquire yeast telomere sequences. This indicated that the yeast was able to add sequences de novo to chromosomes, says Carol Greider of Cold Spring Harbor Laboratory, who was then working with Blackburn as a graduate student. To find the enzyme that might be tacking on these additional sequences, Greider and Blackburn again turned to the ciliate _Tetrahymena_ because, with its horde of telomeres, it was likely to be a much richer source of the enzyme than yeast. By the mid-1980s they had succeeded. "We found telomerase", Greider says, "but it was hard to believe at first because it was such an unusual polymerase." A most unusual enzyme Indeed, telomerase is unlikely nearly all other enzymes researchers have studied. It contains an essential RNA component in addition to the expected protein. Subsequent work by the Blackburn group showed that the RNA serves as the template for the synthesizing the telomere repeats - and it may even play a key role in the enzyme's chemical receptivity. Mutations in the RNA alter the enzymatic properties of telomerase, says Blackburn, a finding that led the group to speculate that "the RNA and the protein may be collaborating in forming the active site [the part of the enzyme that carries out the catalysis]". Because RNA is thought to have played a dual role as both enzyme and repository of genetic information in a primordial "RNA world", the collaboration of an RNA with a protein in the telomerase suggests to Blackburn that the enzyme might be an intermediate in the evolution from the RNA world to the current world of DNA and protein. Blackburn is the first to point out that that intriguing idea is strictly speculative. But it's not the only reason telomerase is attracting attention: It may play a role in overriding the processes that determine the proliferative life span of mammalian cells. Researchers have known since the work of Leonard Hayflick in the 1960s that normal cells have a limited life span in culture, with cells taken from younger individuals dividing more times before they becom senescent and die than do cells from older individuals. An indication that the telomeres might set the cellular clock came in the Blackburn group's experiments on the effect of mutations in the RNA of _Tetrahymena_ telomerase. "One particular mutation changed the activity so badly that the telomeres got shorter and shorter until the cells died", she says. The presence of telomerase in the ciliates may thus make these unicellular organisms immortal by allowing them to keep replenishing the DNA that would otherwise be lost every time the cells divide. But as Hayflick's experiments showed, normal mammalian cells are not immortal. And several research teams have found evidence that their limited life span may be due to telomere shortening. For example, Howard Cooke of the Medical Research Council's Human Genetics Unit as Western General Hospital in Edinburgh, Scotland, and teams led by Titia de Lange of Rockefeller University and by Robin Allshire and Nicholas Hastie, also in Edinburgh, found that human sperm cells - whose clocks have just started ticking - have much longer telomeres than ordinary tissue cells that have gone through multiple rounds of cellular division. And work by Greider, Harley, and their colleagues showed direetly that telomeres shorten every time normal cells divide in culture. Greider notes that the investigators were initially surprised when they saw this shortening, "because that wasn't seen in _Tetrahymena_ and the other ciliates". The most likely explanation for this, she says, is that normal human cells, unlike those of the ciliates, seem to lack a functional telomerase that can replace the chromosome ends lsot to incomplete DNA replication. Exactly what might cause cells to stop dividing and die when their telomeres get too short is unclear, but recent results point to a couple of possibilities. Zakian and her graduate student Lisa Sandell, for example, found that removing a single telomere from a yeast chromosome temporarily blocked cell division, and that this effect depends on the activity of the RAD9 gene. Beeause RAD9 halts the growth of cells whose DNA has been damaged, allowing for repairs, the result suggests that telomeres may be part of the cell's damage sensing system. Other groups, meanwhile, have evidence that loss of telomere DNA may lead to activation of the p53 tumor suppressor gene, which serves the same function in mammalian cells as RAD9 does in yeast: arresting cell growth in response to DNA damage. Another possibility is suggested by work from researchers, including Zakian and Daniel Gottschling of the University of Chicago, who have shown that intact telomeres somehow repress the activity of nearby genes. Some of these genes might trigger cell death once the telomeres becom short enough to allow their expression. Immortal cells But none of this means that it's good for mammalian cells to preserve their telomeres. Indeed, just the opposite may be true. Evidence is now accumulating that the presence of telomerase in cells that normally lack it may contribute to the uncontrolled cell growth of cancer. One hint that this may be the case is the fact that the human enzyme was first detected, by Gregg Morin of the University of California, Davis, in HeLa cells, a cultured cell line originally derived from a human cervical cancer. Following up on that 1989 observation, Harley, along with McMaster's Bacchetti and Cold Spring Harbor's Greider, decided to see whether telomerase activation might allow cultured cells to escape the normal limits on their growth and become "immortalized". And indeed it did. When the researchers tried to mimic the tumor-forming process by introducing oncogenes from simian virus 40 or an adenovirus into cultured cells, they found that telomerase activation was a good predictor of immortality. The oncogenes stimulated cell growth, as expected, and most cells' telomeres continued to shorten with each division until the cells died. But some survived and continued to divide - and those cells, Harley says, had an active telomerase, which presumably stabilized their telomeres. Exactly what allowed the cells to make telomerase is unclear, although the Harley-Bacchetti group detected numerous abnormal chromosomes in cells that had extremely short telomeres. These chromosomal abnormalities, which are similar to those commonly seen in cancer cells, may have led to mutations in the telomerase gene and in other genes that contribute to tumor malignancy. "When cells lose telomeric DNA, it may open the window for all kinds of horrible things to happen", says Zakian, who has noted similar chromosome abnormalities in her yeast experiments. Most recently, Harley, Bacchetti, and their colleagues have turned their attention to cells taken directly from cancerous tumors. They reported in the 14 April _Proceedings of the National Academy of Sciences_ tht telomerase is active in ovarian cancer cells, although not in normal ovarian tissue. The reearchers have since gone on to survey telomerase activity in a variety of additional human cancers, and the preliminary results look promising. Those findings, together with the signs that the enzyme is not active in normal cells, make it an important new target for cancer drugs. "We think [telomerase] will be a specific and probably universal target in tumorigenesis", says Harley, who last year moved to the biotech firm Geron Inc., in Menlo Park, California, to work on developing inhibitors of the enzyme. The hope is that if telomerase activity can be blocked, cancer cells won't be able to maintain adequate telomere length and will die. This may be easier said than done, however. For one thing, researchers still have much to learn about telomerase. So far, they have not been able to isolate the protein portion from any organism, and they have obtained the RNA part only from ciliates. That may soon change, however. Both Blackburn and Gottschling have described candidates for yeast telomerase RNAs at meetings. And a yeast gene identified by Szostak and Victoria Lundblad, who's now at Baylor College of Medicine in Houston, may encode the protein component of yeast telomerase. Greider's group also has a candidate telomerase protein, although in this case from a ciliate. Having the complete enzyme may aid researchers in designing inhibitors, but whether they will actually kill cancer cells remains to be established. Blackburn finds, for example, that some yeast cells are able to survive without telomerase. "There is life without telomerase", as she puts it. That may be true for cells, but for many researchers, the enzyme - along with the telomeres it synthesizes - is now an essential part of their scientific lives. -Jean Marx SCIENCE - VOL. 266 (387-388) - 21 OCTOBER 1994 CHROMOSOME END GAMES Thomas R. Cech The linear chromosomes typical of higher organisms have an obvious feature not found in circular bacterial chromosomes: They have ends. The DNA double helix in the chromosome interior is replicated by DNA polymerase. The polymerase does not start DNA chains de novo, but always reaches to one side and extends the end of a preexistent primer strand bound to the template. Such an enzyme is frustrated at the chromosomal end, or telomere. If you are already at the end of a line and reach outward, there is nothing there to extend. The solution to this problem is telomerase, a telomere-extending enzyme that until recently had been subject to molecular analysis only in ciliated protozoa. This situation has now changed with discoveries of telomerase enzymes in yeast cells, one of which was reported in this issue of _Science_ (1). Telomerase was first described a decade ago by Carol Greider and Elizabeth Blackburn in _Tetrahymena_, a single-celled pond organism with an unusually large nmber of nuclear DNA molecules and therefore many telomeres (2). Telomerase is not the usual protein enzyme but is instead a ribonucleoprotein. Its RNA subunit includes a 5'-CAACCC-3' sequence that serves as a template for the addition of 5'-GGGTTG-3' repeats to chromosome ends (3) (see figure). As in _Tetrahymena_, most other eukaryotic chromomsomes terminate in repeats of a short DNA sequence with one strand rich in G (guanine) bases, so it seemed likely that telomerase would be the key to telomere replication in general. Indeed, the _Tetrahymena_ telomerase served as the springboard for cloning and sequencing the telomerase RNA subunits from a number of other ciliated protozoa (4). But the RNA turned out to have a fast evolutionary clock: Outside the template region its sequence diverged rapidly from species to species. Thus, although the activity of the enzyme could be detected in diverse cells, including human cells (5), isolation of any molecular component of telomerase remained restricted to the ciliated protozoa. The announcement by Singer and Gottschling of the finding of the gene for telomerase RNA in the yeast _Saccharomyces cerevisiae_ (1), a tractable system for genetic manipulation, has therefore been enthusiastically received. The isolation of the telomerase RNA gene from another yeast, _Kluveromyces lactis_, has recently been reported (6), so there should soon be two new telomerases to study and compare. There is little doubt that Singer and Gottschling have now found the yeast telomerase and cloned its RNA component. Knocking out the gene caused progressive shortening of yeast telomeres by about 3 base pairs per generation. This shrinking telomere phenotype is that expected for inactivation of telomerase, which compensates for the inability of DNA polymerase to complete replication at chromosome ends. Furthermore, the putative telomerase RNA contained a sequence perfectly complementary to the 13-nucleotide sequence frequently found at newly created telomeres in yeast cells (7). When Singer and Gottschling altered two bases in the proposed template sequence of their telomerase RNA gene and reintroduced it into yeast, the altered DNA sequence was incorporated at a telomere. Thus, their isolated gene encodes the RNA responsible for templating telomere synthesis in yeast cells. The stategy by which Singer and Gottschling unearthed the yeast telomerase RNA already provides some new insights into telomerase function. They engineered a system in which two reporter genes were placed near telomeres. Gene expression is repressed near yeast telomeres, presumably because the genes are buried in a higher order structure involving specific chromosomal proteins (8). Thus, the test genes were initially in the "off" state. These cells were transformed with a yeast complementary DNA expression library: a vast array of plasmids containing unidentified yeast genes, a different one in each cell. The hypothesis was that the production of an abnormal abundance of one component of the multicomponent telomeric complex could titrate another component and disrupt normal telomeric chromatin assembly. A cell containing such a plasmid would be relieved of telomere-proximal gene repression. So why should the telomerase enzyme turn up in such a screen? An intriguing possibility: One of the molecules binding to telomerase RNA may also serve as a component of the telomeric chromatin complex. It is even coneivable that the entire telomerase ribonucleoprotein is a component of the complex. This would not be tenable for all the telomeres in certain ciliated protozoa, because telomeres outnumber telomerase 100 to 1, but it may be possible in yeast cells, where telomerase seems moderately abundant and needs to serve only 16 chromosomes. An alternative hypothesis to explain the relief of gene repression by overexpression of telomerase RNA requires an indirect chain of events. Because the telomeric DNA shrinks when telomerase RNA is oversupplied, binding sites for telomeric proteins may be depleted and the inhibitory telomeric complex may thereby be disrupted. Particularly exciting are the nine plasmids isolated by Singer and Gottschling that do not encode telomerase RNA, but also pass their screen and are therefore implicated in telomere function. Of corse, these could be general chromatin proteins or other DNA binders, including molecules that do not even interact with telomeres unless overexpressed. A major breakthrough would result if this collection included another telomerase subunit - a protein subunit. Although there is evidence for such protein components and even some plausible candidates (9), no protein component of telomerase has been unequivocally identified in any organism. Will protein components of telomerase really be very important? After all, we have the ribonuclease P paradigm: A ribonucleoprotein enzyme can have a catalytic RNA subunit and an accssory protein (10). However, I think it unlikely that telomerase is a ribozyme. Compared to RNAs known or thought to participate directly in catalysis (such as group I and II introns, ribonuclease P, U6 small nuclear RNA, and ribosomal RNA), telomerase RNA appears to be less structured and to have few conserved nucleotides. Thus, catalysis of DNA expression is likely to occur in a protein active site. Yet the RNA may serve more than just a template function. For example, it may organize a number of proteins into an active complex. These new discoveries (1,6) provide great optimism that telomerase RNA can now be identified in larger eukaryotes, including humans. Because activation of telomerase correlates with oncogenic transformation, both in cell culture and in human tumors, telomerase inhibitors might have anticancer activity (11). Having the human telomerase in hand would facilitate development of such pharmaceutical agents. Given telomerase RNA's fast evolutionary clock, the leap from yeast to human cells may be difficult. Meanwhile, the awesome power of yeast genetics, which has been so successful in unravelling secrets of another ribonucleoprotein machine, the spliceosome (12), will be unleashed on telomerase. Yeast genetics seems ideally suited to reveal additional functions of telomerase RNA and to identify protein components of this essential enzyme. References 1. M.S. Singer and D.E. Gottschling, _Science_ 266, 404 (1994). 2. C.W. Greider and E.H. Blackburn, _Cell_ 43, 405 (1985). 3. ___ _Nature_ 337, 331 (1989). 4. D.P. Romero and E.H. Blackburn, _Cell_ 67, 343 (1991); D. Shippen-Lentz and E.H. Blackburn, _Science_ 247, 546 (1990); J. Lingner, L.L. Hendrick, T.R. Cech, _Genes Dev._ 8, 1984 (1994). 5. G. Morin, _Cell_ 59, 521 (199). 6. E.H. Blackburn and M.J. McEachern, lecture presented at Keystone Symposium on the Eukaryotic Nucleus (1994). 7. K.M. Kramer and J.E. Haber, _Genes Dev._ 7, 2345 (1993). 8. D.E. Gottschling, O.M. Aparicio, B.L. Billington, V.A. Zakian, _Cell_ 63, 751 (1990); D.E. Gottschling, _Proc. Natl. Acad. Sci. U.S.A._ 89, 4062 (1992). 9. V. Lundblad and J.W. Szostak, _Cell_ 57, 633 (1989). 10. C. Guerrier-Takada, K. Gardner, T. Marsh, N. Pace, S. Altman, _ibid._ 35, 849 (1983). 11. T. de Lange, _Proc. Natl. Acad. Sci. U.S.A._ 91. 2882 (1994); J. Marx, _Science_ 265, 1656 (1994). 12. C. Guthrie, _Science_ 253, 157 (1991). "The author is in the Howard Hughes Medical Institute, Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO 80309-0215, USA." -- -mitch http://student.uq.edu.au/~ma157727 http://www.phantom.com/~slowdog for S.314 data