Telomerase and its implications for aging and cancer - Lasker Foundation

Elizabeth H. Blackburn

University of California, San Francisco

Carol W. Greider

Johns Hopkins University, School of Medicine

Jack W. Szostak

Harvard Medical School

For the prediction and discovery of telomerase, a remarkable RNA-containing enzyme that synthesizes the ends of chromosomes, protecting them and maintaining the integrity of the genome.

The 2006 Albert Lasker Award for Basic Medical Research honors three scientists who predicted and discovered telomerase, an enzyme that replenishes the ends of chromosomes. In so doing, they unearthed a biochemical reaction that guards cells against chromosome loss and identified the molecular machinery that performs this feat. The work resolved perplexing observations about chromosome termini and explained how cells copy their DNA extremities.

In the 1930s, scientists surmised that protective caps — telomeres — ensure the propagation of chromosomes during cell division and prevent them from inappropriately melding with one another. The physical nature of these structures — and how they are constructed — eluded researchers until Elizabeth Blackburn, Carol Greider, and Jack Szostak performed their groundbreaking investigations in the late 1970s and 1980s. Blackburn showed that simple repeated DNA sequences comprise chromosome ends and, with Szostak, established that these repeats stabilize chromosomes inside cells. Szostak and Blackburn predicted the existence of an enzyme that would add the sequences to chromosome termini.

Greider and Blackburn then tracked down this enzyme — telomerase — and determined that each organism’s telomerase contains an RNA component that serves as a template for the creature’s particular telomere DNA repeat sequence. Szostak found that budding yeast unable to perform the telomerase reaction lose their telomeres — and chromosomes — over multiple generations. Eventually, the organisms stop dividing. In addition to providing insight into how chromosome ends are maintained, Blackburn’s, Greider’s, and Szostak’s work laid the foundation for studies that have linked telomerase and telomeres to human cancer and age-related conditions.

The beginning of the ends

In the 1930s, Barbara McClintock (Lasker 1981) and Hermann Muller independently inferred that the natural termini of chromosomes display special characteristics. Unlike ends generated by DNA breakage, they don’t fuse with each other. Furthermore, only chromosomal fragments containing intact ends persist when a cell duplicates. A distinct structure must seal chromosomes and confer these properties, Muller reasoned. He dubbed chromosome termini “telomeres” from the Greek telos for end and meros for part or segment. However, no one knew what made telomeres different from randomly generated ends.

A second telomere-related conundrum arose after researchers deciphered how eukaryotic cells — those with nuclei — copy DNA. The enzyme that performs this reaction should be unable to fully replenish linear DNA due to a peculiarity of its mechanism; each round of replication should generate a molecule missing a few building blocks, called nucleotides, on the DNA’s end. As a result, linear chromosomes — which house genes in eukaryotes — would shorten every time a cell divides. In 1972, James Watson (Lasker 1960) speculated that organisms with linear chromosomes need a strategy to maintain chromosome tips, a theory that became known as the “end-replication problem.” In parallel, Alexey Olovnikov suggested that the gradual loss of chromosome ends would lead to cellular senescence, a dormant state in which cells remain alive but can no longer divide or perform their normal functions. Although scientists discussed possible solutions to these problems, they did not have ways to test their ideas.

The ends in sight

In the late 1970s, Blackburn wanted to determine the sequence of DNA at the ends of a eukaryotic chromosome. Joseph Gall, then at Yale University, had found that the ciliated protozoan Tetrahymena thermophila contains many DNA minichromosomes. Because the molecules are small but abundant, the number of ends relative to the rest of the DNA is large. This feature of Tetrahymena allowed Blackburn, working in Gall’s lab as a postdoctoral fellow, to gather enough ends to sequence. Each was composed of a six-nucleotide sequence (CCCCAA) that was repeated 20–70 times. Similar sequences turned out to reside in other ciliates, but no one knew whether this odd feature appeared in distantly related organisms.

In 1980, Blackburn, by then running her own lab at the University of California, Berkeley, presented her work at the Nucleic Acids Gordon Research Conference. After her talk, she spoke with Jack Szostak, a yeast geneticist from Harvard Medical School. They decided to add the Tetrahymena repeats to the ends of linear DNAs and test whether the resulting DNA would persist in budding yeast. Szostak knew that non-chromosomal linear DNAs in yeast normally insert themselves into chromosomes or are destroyed by cellular enzymes, presumably because they behave as if they result from random fractures. The Tetrahymena sequences provided the first hope for yeast to retain such linear DNAs. The experiment worked, despite the vast evolutionary distance between budding yeast and Tetrahymena. The Tetrahymena telomeres protected the linear yeast DNA, allowing it to pass reliably from one generation to the next.

The researchers then identified short, distinctive repeats on the ends of normal yeast chromosomes and showed that this yeast telomeric sequence was tacked on to Tetrahymena ends that were present on linear DNA in yeast. Because yeast added its characteristic sequence to Tetrahymena telomeres, telomeres must not serve as templates for additional telomeric sequences. This finding and the varied number of repeats led Blackburn and Szostak to speculate that an enzyme adds telomeric sequences to chromosome ends. Such an act would replenish the genetic material predicted to be whittled away by DNA replication. This idea differed radically from other suggestions scientists had proposed to solve the end-replication problem.

The means to the ends

Blackburn, soon joined by Greider, who was then a graduate student, started seeking the hypothetical enzyme by looking for a substance that could affix telomeric repeats to chromosome ends in the test tube. Because Tetrahymena contained so many telomeres, the organism should provide a rich source of enzymes that act on them, the researchers reasoned. They added the contents of Tetrahymena cells to a mixture of radioactive nucleotides and small DNA pieces composed of the Tetrahymena telomere repeats, hoping to see the tagged nucleotides attach to the repeats. On Christmas Day 1984, Greider discovered that the Tetrahymena contents generated precisely the DNA pattern predicted for an enzyme that added the six-nucleotide repeats one building block at a time. Blackburn and Greider named the enzyme telomere terminal transferase.

Next, they wanted to figure out exactly how the telomere sequence was determined. They postulated that each organism’s enzyme contained an RNA or DNA component that could serve as a template. An RNA-destroying enzyme obliterated the telomere terminal transferase activity, so Blackburn and Greider concluded that an RNA must play a crucial role. In a tour de force of biochemistry, Greider purified the enzyme, which they now called telomerase, and showed that it contained both an RNA and a protein subunit. In her own lab at Cold Spring Harbor Laboratory, she completed this work by isolating the RNA-encoding gene. It indeed carried a sequence that could specify the Tetrahymena telomere repeats. Furthermore, cleaving that sequence demolished telomerase activity. To prove that the internal template determines the sequence of the telomeric repeats, Blackburn altered the crucial sequence in the RNA component of telomerase; this perturbation resulted in production of telomeres that correspond to the new sequence. Later, Tom Cech (Lasker 1988) purified the protein portion of telomerase, which adds nucleotides one by one to the chromosome ends, according to instructions from the RNA component.

In the meantime, Szostak and his postdoctoral fellow Victoria Lundblad established that the inability to restore telomeres imperils the cell. They had been seeking yeast mutants that could not properly elongate telomeres. This scheme should identify genes that are crucial for telomerase function, they reasoned. Telomeres in such mutants would shrink, Szostak and Lundblad predicted, and strains harboring such defects would lose their chromosomes over many generations. At first, the yeast would grow normally, but as the genetic material disappeared, the microbes would stop dividing. The researchers found such a strain — and named the gene responsible for the defect EST1 for “ever shorter telomeres.” The approach subsequently led to the discovery of other proteins required for telomere stability, including telomerase’s core protein. Furthermore, the finding provided the first experimental support for the end-replication problem: As predicted, the inability to replenish telomeres caused the structures to dwindle as cells reproduced. Moreover, the work implied that cells unable to solve the end-replication problem eventually senesce.

All’s well that ends well

The work by Blackburn, Greider, and Szostak set the stage for discoveries about the role of telomerase in human cancer and aging. Like their Tetrahymena and yeast counterparts, human telomeres are composed of a particular simple DNA sequence, repeated various numbers of times. Sperm and eggs manufacture telomerase, but most adult cells don’t — and telomeres in most adult cells are shorter than those in sperm and eggs. Telomere attrition, at least in cells grown outside the body, leads to senescence.

Approximately 85 to 90 percent of human cancers reactivate telomerase (the rest maintain their telomeres through an alternative mechanism) and strong evidence suggests that the enzyme renders these cells able to proliferate uncontrollably by continually refreshing their telomeres. For example, adding the enzyme to certain human cells grown in culture dishes renders the cells immortal. Conversely, blocking the enzyme’s action in lab-grown cancer cells can inhibit their growth or kill them. Scientists are pursuing compounds that thwart telomerase as a potential strategy for fighting cancer. Several clinical trials of such drugs are now under way.

Along similar lines, telomere erosion during a person’s lifetime could curtail cell survival, thereby promoting age-related ailments. Evidence supporting this notion comes from studies of the rare human disease dyskeratosis congenita. One form of this illness arises from genetic defects in the RNA component of telomerase. Short telomeres limit the ability of certain tissues to replace themselves. As a result, the disease generates age-like conditions: It wipes out affected individuals’ bone marrow, predisposes them to a variety of human cancers, and gives them splotchy skin, ratty fingernails, and prematurely gray hair. The work on dyskeratosis congenita demonstrates that withered telomeres can accelerate physical deterioration.

Blackburn, Szostak, and Greider pursued basic questions of cell biology and enzymology to unveil mysteries that have huge implications for human health. The impact of their work is certain to extend long into the future.

by Evelyn Strauss

*Figure. Visualizing chromosome tips. Chromosomes (blue) carry telomeres (yellow) at their ends. The technique used for generating this image is a form of in situ hybridization, which was developed by Joseph Gall, this year’s winner of the Lasker Award for Special Achievement in Medical Science. To detect the telomeres, sequences that match the telomere repeats were tagged with fluorescent yellow molecules and applied to the chromosomes. The chromosomes were stained blue with a dye that binds DNA. [Credit: Peter M. Lansdorp, BC Cancer Research Centre, Vancouver, Canada]

Key publications of Elizabeth Blackburn

Blackburn, E.H. and Gall, J.G. (1978). A tandemly repeated sequence at the termini of the extrachromosomal ribosomal RNA genes in Tetrahymena. J. Mol. Biol. 120, 33–53.

Greider, C.W. and Blackburn, E.H. (1985). Identification of a specific telomere terminal transferase activity in Tetrahymena extracts. Cell. 43, 405–413.

Greider, C.W. and Blackburn, E.H (1987). A telomeric sequence in the RNA of Tetrahymena telomerase required for telomere repeat synthesis. Nature. 337, 331–337.

Yu, G.L, Bradley, J.D., Attardi, L.D., and Blackburn, E.H. (1990). In vivo alteration of telomere sequences and senescence caused by mutated Tetrahymena telomerase RNAs. Nature 344, 126–132.

McEachern, M.J. and Blackburn, E.H. (1995). Runaway telomere elongation cause by telomerase RNA mutations. Nature. 376, 403–409.

Kim, M.M., Rivera, M.A., Botchkina, I.L., Shalaby, R., Thor, A.D. and Blackburn, E.H. (2001). A low threshold level of expression of mutant-template telomerase RNA is sufficient to inhibit tumor cell growth. Proc. Natl. Acad. Sci. USA. 98, 7982–7987.

Key publications of Carol Greider

Greider, C.W. and Blackburn, E.H. (1985). Identification of a specific telomere terminal transferase activity in Tetrahymena extracts. Cell. 43, 405–413.

Greider, C.W. and Blackburn, E.H. (1987). The telomere terminal transferase of Tetrahymena is a ribonucleoprotein enzyme with two kinds of primer specificity. Cell. 51, 887–898.

Greider, C.W. and Blackburn, E.H (1987). A telomeric sequence in the RNA of Tetrahymena telomerase required for telomere repeat synthesis. Nature. 337, 331-337.

Allsopp, R.C., Vaziri, H., Patterson, C., Goldstein, S., Younglai, E.V., Futcher, C.W., Greider, C.W., and Harley, C.B. (1992). Telomere length predicts the replicative capacity of human fibroblasts. Proc. Natl. Acad. Sci. USA. 89, 10114–10118.

Prowse, K.R., Avilion, A.A., and Greider, C.W. (1993). Identification of a nonprocessive telomerase activity from mouse cells. Proc. Natl. Acad. Sci. USA. 90, 1493–1497.

Rudolph, K.L., Chang, S., Lee, H.W., Blasco, M., Gottlieb, G., Greider, C.W., and DePinho, R.A. (1999). Longevity, stress response, and cancer in aging telomerase deficient mice. Cell. 96, 701–716.

Key publications of Jack Szostak

Szostak, J.W. and Blackburn, E.H. (1982). Cloning yeast telomeres on linear plasmid vectors. Cell. 29, 245–255.

Murray, A.W. and Szostak, J.W. (1983). Construction of artificial chromosomes in yeast. Nature. 305, 189–193.

Blackburn, E.H. and Szostak, J.W. (1984). The molecular structure of centromeres and telomeres. Ann. Rev. Biochem. 53, 163–194.

Shampay, J., Szostak, J.W., and Blackburn, E.H. (1984). DNA sequences of telomeres maintained in yeast. Nature. 310, 154–157.

Dunn, B.L., Szauter, P., Pardue, M-L., Szostak, J.W. (1984). Transfer of telomere-adjacent sequences to linear plasmids by recombination. Cell. 39, 191–201.

Lundblad V. and Szostak, J.W. (1989). A mutant with a defect in telomere maintenance leads to senescence in yeast. Cell. 57, 633–643.

Award presentation by Joseph Goldstein

The discovery of telomerase centers around four principal characters — a single-cell protozoan that swims in freshwater ponds, propelled by hairlike projections called cilia; a Tasmanian-born devourer of Amanda Cross mystery novels; a triathlete and competitive vaulter who performs handstands on the back of a speeding horse; and a 53-year-old virgin prizewinner.

So what is telomerase and why is it Lasker Prize-worthy? As you all know, life depends on the genetic instructions carried in the DNA of the genome.

In the human genome, our 25,000 genes are distributed over 23 different pairs of chromosomes. Each chromosome consists of a single, enormously long DNA molecule that encodes a linear array of many genes. The tips of each chromosome terminate in a specialized structure called the telomere. Telomeres act like protective shields in much the same way as plastic tips safeguard the ends of shoelaces; they stop chromosomes from fraying. The crucial role of telomeres in maintaining the integrity of chromosomes was first recognized in the early 1940s by two famous Nobel Prize-winning geneticists, Herman J. Muller and Barbara McClintock. Studying different organisms under the microscope, Muller with fruit flies and McClintock with corn observed that when chromosomes lack telomeres they stick to one another, producing catastrophic fusions and other abnormal structural changes that threaten cell replication and survival. Muller coined the term “telomere” from the Greek word for “end” (telos). His choice of telos may have been more apt than he intended; after his and McClintock’s work in the early 1940s, all research on telomeres came to an abrupt end and did not resume for another 30 years.

Then, in 1972, James Watson of double helix fame and Alexey Olovnikov, a young Russian scientist with no fame, independently made an important theoretical advance that reignited interest in telomeres. Watson and Olovnikov noted that the polymerase enzymes that normally replicate DNA every time a cell divides are not able to copy the chromosomes all the way to the very tip, leaving a small region at the end of each telomere uncopied, much like a tape recorder cannot play the last few centimeters of tape in a cassette. If cells had no way of compensating for this quirk in the replication machinery, chromosomes would shorten with each round of cell division, and ultimately there would be an erosion of telomeric DNA such that the telomeres would no longer perform their protective function. Watson’s and Olovnikov’s theory, which became known as the “end-replication problem,” predicted that cells possess a special strategy to maintain their telomeres during normal DNA replication.

And that special strategy turned out to be the enzyme telomerase. In 1975, Elizabeth Blackburn, a native of Tasmania and a newly minted PhD from Fred Sanger’s laboratory at the MRC in Cambridge, crossed the Atlantic to begin a postdoctoral fellowship with Joseph Gall, then at Yale University. Gall was — and still is — a renowned cell biologist whose career you’ll hear about in a few minutes when he receives the Lasker Special Achievement Award. As an avid reader of Amanda Cross mystery novels, Blackburn was anxious to solve a mystery of her own.

She came to Gall’s lab to work on an organism with highly unusual chromosomes — a ciliated protozoan called Tetrahymena. Unlike typical eukaryotic organisms, Tetrahymena has two nuclei — a micronucleus that contains normal chromosomes and a macronucleus whose chromosomes are fragmented into thousands of small pieces of DNA that all encode the same ribosomal RNA gene. Because of the high abundance of this one extrachromosomal gene, Blackburn and Gall were able to purify the molecule and determine the DNA sequence at its tips. The sequence turned out to be unique — a short simple sequence of six nucleotides, TTGGGG, which was repeated over and over 50 times at both ends of the molecule. This type of tandem repeat had never been seen before, and its discovery immediately raised the question as to whether it was a peculiar property of an unusual extrachromosomal gene in a ciliated organism far removed from the main line of eukaryotic evolution — or was it an authentic telomere sequence that would have universal relevance to the chromosomes of humans and other higher organisms? Blackburn no longer had time for Amanda Cross mysteries; she her own mystery to solve.

The first clue came in 1982 when Blackburn began collaborating with Jack Szostak, a yeast geneticist and molecular biologist who was just starting his own lab at Harvard Medical School. Blackburn, too, was just starting her own lab at the University of California in Berkeley. At that point in time — 1982 — cloning of recombinant DNA molecules was done in bacteria with circular plasmids that held only about one or two gene’s worth of DNA. Szostak had started a bold project aimed to construct artificial chromosomes that would enable scientists to clone a large cluster of human genes — 10 to 20 — on a single linear molecule of DNA in yeast cells. The first versions of Szostak’s artificial chromosomes were unstable and did not propagate in the yeast cells, presumably because they lacked telomeres.

When Szostak heard about Blackburn’s Tetrahymena repeat sequences, he proposed testing whether they would function as telomeres in his yeast system. This was a stunningly novel experiment, and the results were spectacular. The linear yeast plasmids containing the Tetrahymena repeat ends replicated in stable fashion. Szostak and Blackburn had now developed the first functional assay for telomeres. This experiment also formed the basis for Szostak’s later work on the creation of the first yeast artificial chromosomes — the famous YACs that played a key role in the Human Genome Project by enabling scientists to clone long pieces of human DNA that were used for sequencing.

When Blackburn and Szostak sequenced their newly manufactured yeast plasmid containing the Tetrahymena repeat sequence, the results were totally unexpected: The replicated plasmid was longer than it should have been; the yeast cells had actually added many copies of a new type of repeat sequence onto the end of the Tetrahymena repeat sequence. This discovery led Szostak and Blackburn to predict that the yeast cells contained a telomere-synthesizing enzyme — soon to be called telomerase. The hunt for the telomerase had now begun. Blackburn took a biochemical approach, and Szostak a genetic approach.

Blackburn found the biochemical search to be exceptionally difficult. She began with crude extracts of nuclei that contained many other enzymatic activities unrelated to telomerase, including nucleases that degrade any newly made nucleic acid. She also had to worry that any telomerase activity she saw was an aberrant result caused by one of the DNA polymerases that replicate the middle of the chromosomes. It was like looking for a needle in a haystack. After six months of looking, Blackburn never found the needle, but instead she turned up the farmer’s daughter — Carol Greider.

In 1984, Greider entered graduate school at Berkeley. Trained as an equestrian vaulter, Greider proved to be the white knight in shining biochemical armor who galloped into Blackburn’s cold room and four years later emerged with the mythical telomerase in hand — purified and characterized. The key to Greider’s success was the development of an innovative assay, not to mention a lot of hard work and ice-cold fingers. The telomerase that Greider and Blackburn had discovered was more interesting than anyone could have imagined.

To make a long story short, telomerase is a multi-subunit enzyme composed of both RNA and protein components. The most unique component — the one that Greider purified and cloned — is an RNA molecule that contains a built-in template that ensures that the catalytic protein component of the enzyme adds the correct repeat sequence to the ends of the chromosome. The catalytic component of telomerase, a reverse transcriptase-like enzyme, was subsequently purified and cloned by Tom Cech and colleagues. Telomerase also contains several regulator proteins essential for its function.

Meanwhile, Jack Szostak was not resting on his laurels at Harvard. Together with a talented postdoctoral fellow, Victoria Lundblad, Szostak had embarked on a clever genetic strategy designed to detect mutants in yeast that would be defective in any essential component of the putative telomerase enzyme and its regulators. Their first mutant, called EST1 for “ever shorter telomeres,” had a dramatic phenotype. Telomeric DNA gradually disappeared, chromosomes became shorter with each succeeding cell division, and the cells underwent premature aging as their telomere reserve was depleted. These findings provided the first experimental evidence linking the length of telomeres to the aging of cells — a subject I’ll say more about in a moment.

Later work by Lundblad showed that the protein lacking in the EST1 mutant was one of the accessory regulator proteins in the telomerase complex. Another EST mutant, EST2, actually lacked the catalytic component of telomerase — the same protein that Tom Cech had purified, providing a neat genetic validation of the complex biochemistry pioneered by Greider and Blackburn.

The basic discoveries of Blackburn, Szostak, and Greider stimulated hundreds of scientists to enter the field in 1990s, and this explosion of research has led to a new model of how the life span of normal cells is regulated and how this regulation goes astray in cancer. As cells in the body grow old, telomeres progressively shorten because normal cells contain only minute amounts of telomerase. Once the telomeres shorten beyond a critical point, aging cells undergo a crisis, and the vast majority of them die. The few cells that survive are the ones that have activated their telomerase. These surviving cells become immortalized, divide indefinitely, and are now poised to become malignant. More than 85 percent of all human cancers have elevated telomerase that propels their growth. Inhibition of telomerase provides an exciting new drug target for cancer therapy, which many pharmaceutical companies are actively pursuing.

Telomerase strikes at the very core of our longevity and our mortality. Its popularity has inspired thousands of newspaper and magazine articles as well as several books, including Merchants of Immortality by Stephen Hall, an engaging science writer who is here with us today.

I can’t resist ending this talk without a telomeric ending that contains a few repeats. Remember at the beginning when I mentioned those four principal characters. I’ve already told you about the ciliated protozoan in Joe Gall’s lab that got the ball rolling, I’ve told you about Liz Blackburn and her obsession to solve scientific mysteries, and I’ve told you about Carol Greider and her handstands in the cold room. But I didn’t explain the 53-year-old virgin prizewinner. In the past 10 years, telomerase has been honored with many prizes — all of them going to Blackburn alone or Blackburn and Greider. This year’s Basic Lasker Award is the first of these telomerase prizes that Jack Szostak shares with Liz Blackburn and Carol Greider.

To be completely honest, Jack is not really a virgin prizewinner. He has received many distinguished prizes for his other research, not involving telomerase. When Jack left the telomerase field 17 years ago, he went on to pioneer several other bold endeavors — the most recent involving an attempt to unravel the origins of life in a test tube by mixing together three elements that existed in the Earth’s ancient chemical soup 4 billion years ago. The goal is to get these three primordial elements — RNA, fatty acids, and a special type of clay — to assemble into a so-called “proto-cell” that will replicate genetic material, form cell membranes, and divide into daughter cells — all in a test tube. If Jack is successful, the proto-cell will speak to the origins of life in much the same way that telomerase speaks to our longevity and our mortality.

This discovery of telomerase is a great story in the history of biomedical science. Congratulations to all three of you — Liz, Carol, and Jack.

Acceptance remarks

Interview with Elizabeth Blackburn, Carol Greider, and Jack Szostak