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Telomeres as the Clock of Cell Aging and Immortalization

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  "But the lengthening of the thread of life itself, and the postponement for a time of that death which gradually steals on by natural dissolution and the decay of age, is as subject which no physician has handled in proportion to its dignity."  
 

Francis Bacon
The Advancement
of Learning

 

My own research in the field of molecular and cellular gerontology began in 1982 in the laboratory of Dr. Samuel Goldstein. I telephoned Sam to explain my interest in the biology of aging and discuss the possibilities of joining his lab as a graduate student. I related to Sam that I was considering exploring the mechanisms of aging in a number of ways. I told him I had visited with Joan Smith Sonneborn to potentially join her lab in the study of aging in the microscopic pond water animal paramecia. Sam quickly rejoined, “At the end of the day, maybe you would learn something about the aging of protozoa. Why not study human fibroblast aging? If your interest is in human aging, why not use human cells?”

Sam had devoted much of his scientific career to the study of cellular aging beginning in the 1960s with a landmark study demonstrating the short lifespan of cells from children with the premature aging syndrome known as progeria1. Sam’s point about focusing on human cell aging resonated with me, as did his willingness to take me on as a student. I relate more of the personal details of those early days of aging research in my book The Immortal Cell2.

When I entered the field of cell aging research, there were as many theories of the mechanisms of aging as there were researchers (probably more). Len Hayflick, being a steward of many of these results, summarized in a 1980 article approximately 110 changes that occur in cells as they age in the laboratory, all of which were potentially the cause, and all of them potentially needing reversal to intervene in the process3.

So, with all that complexity, the aging of cells seemed like the inevitable wear-and-tear of everything around us. Cars rust, tires wear out, cars head for the junkyard. Over time, things inevitably head toward disorder. It is, after all, a law of physics, the second law of thermodynamics, right?

Some scientists therefore concluded that the mere conception of stopping or reversing the aging of cells was therefore patently nonsensical. It would be like unscrambling an egg. But, as Len Hayflick pointed out even in those early days, we actually can unscramble an egg. As he stated in a 1990 letter to Science, “An egg can be unscrambled, and the Second Law violated, by feeding it to a hen4.”

Len’s comment was obviously meant to be tongue-in-cheek, nevertheless he was making a subtle and important point. Life itself, inasmuch as it continues to replicate the species, does not drift into disorder. In fact, over the eons, life has slowly evolved into more and more complexity. If the species itself perpetuates bodies from an immortal germ-line lineage of cells, why then does the soma itself live for such a short time? The remarkable, almost jaw-dropping way of thinking about this is that we are made from a lineage of cells with no dead ancestors. The cells that made us could be traced like connecting the dots back in time through our ancestors for thousands, indeed many millions of years all of the way back to the original life forms on the planet billions of years ago. But the collection of cells comprising the body, that is, you and me, are destined to live a mere few decades. As the evolutionist George Williams stated:

  "It is indeed remarkable that after a seemingly miraculous feat of morphogenesis a complex metazoan should be unable to perform the much simpler task of merely maintaining what is already formed5."  

So, the first clue in the path to understand cellular aging was that some cells can escape aging in the process of perpetuating the species. And this probably isn't dependent on the process of sexual reproduction (egg and sperm production) since some advanced animals can perpetuate without sex in a process known as parthenogenesis (virgin birth) with only females egg cells involved.

The second clue was that some mortal somatic cells could find their way to immortality in vitro as well. Even before Len’s pioneering work on cell aging, George Gey at Johns Hopkins University reported that cells from a patient with cervical cancer proliferated abundantly in the laboratory6. Over the years, it became clear that these and many other cancer cells did not necessarily age like the normal cells studied by Hayflick. These first cultured cancer cells isolated by Gey were designated “HeLa” based on her name Henrietta Lacks.

In addition to the immortality Ms. lacks has in the continued culture of her cancer cells, she has attained an additional form of immortality lately in the widespread interest in what happened to these cells in the years following her death thanks to a book titled The Immortal Life of Henrietta Lacks7.

In addition to the immortality of cells derived from cancer, it became apparent that normal cells could be transformed into cancer cells in the laboratory using certain tumor viruses such as papilloma virus (the virus that caused Ms. Lack’s cancer), and a related virus called SV40.

In 1965 Hilary Koprowski’s group showed that SV40 virus could at first extend the cell lifespan of normal human cells in the laboratory dish and then, more rarely, immortalize them into an immortal culture8. The latter cells were said to be an established “line.” An odd thing about these cultures was that the virus could somehow extend the lifespan of the cells, but they still underwent a type of aging called “crisis” later on. And in the cells that had entered crisis there were almost always the presence of chromosomes fused at the ends (the linear ends of the chromosomes being called “telomeres.” So telomere fusions were accompanying this crisis event9.

Another clue was that about one in 10 million cells in the crisis phase in the presence of SV40 virus could find their way to immortality. While on the surface of it, this sounds like a very rare event, when one runs the numbers, it would appear that only one or at most two genes were involved, nothing close to the hundreds of changes catalogued by Len Hayflick.

So, there were clues that the mechanisms of cell aging and immortalization may not be as complex as one would think. But what would those mechanisms be?

Some of the early cell aging research suggested that there must be some kind of molecular clockwork. When Len’s cells were thawed from the freezer, even after many years, they would again begin to proliferate in the dish, and despite the passage of chronological time, they had not aged, but instead took up right where they left off and proliferated the same number of doublings as if they had never been frozen.

To rule out unfrozen chronological time as a cause, Robert Dell’Orco at the Noble Foundation in Oklahoma showed that cells kept in culture in the nondividing (unfrozen) state for extended periods of time still proliferated the same number of doublings as those that had not been so maintained10. So, Dell’Orco concluded that aging human cells were not measuring metabolic time, but instead were “counting” cell divisions. How could a cell do that? It doesn’t have a brain or any known memory mechanism that could count and remember how many times it had divided.

A couple of years later, Woody Wright, working in Len’s laboratory at Stanford performed a difficult test to see where within the cell this clock might be located. In short, Woody swapped the nuclei of young and old cells. Of course, it is within the central core of the cell known as the nucleus that the master blueprint of life called DNA resided. Woody’s experiments suggested that the nucleus of an old cell made a young cell old, and the vice versa. Taking the two observations together, those of Bob Dell’Orco and Woody Wright, experiments of the 1970s indicated that the clock of cell aging must be counting cell divisions and located in the nucleus. But how? No one knew.

My colleague Sam Goldstein believed, somehow intuitively, that the clock may involve changes in the DNA, but not genes themselves. The DNA molecule has regions called “genes” that are the instructions to make things like proteins that do work within the cell. But there are other regions in the DNA, that have, for instance, long stretches of DNA with sequences that are repeated end-on-end that are not technically genes. Sam’s instinct was that there may be progressive changes in these non-coding regions of the DNA that, since they did not encode proteins, functioned instead as a clock. His lab had looked at two of these types of repeated sequences. One, called Eco RI repeats were reported by Sam’s lab to be lost as cells aged11. Another called Inter-Alu were reported by his group to be unstable, popping out of the chromosomes as small circles12. As a graduate student, I attempted to repeat these studies and instead found technical problems with the experiments. Neither of these repeat sequences were really changed in aged cells.

Nevertheless, since Sam’s lab was largely focused on these ideas about DNA repeat loss as a clocking mechanism, it gave me a unique opportunity to think through these models of cell aging clocks. One day, Sam and I were discussing ideas on how this counting mechanism could work. Sam mentioned a theory proposed by a then little known Russian theorist named Alexey Olovnikov.

In the late 1960s, Alexey was a Russian theoretical biologist at the Gamelaya Institute of the Academy of Sciences of the USSR. One day he attended a lecture presented by Alexander Y. Friedenstein, a cell biologist at Moscow University, about Hayflick’s observation that human somatic cells have a finite lifespan. As Olovnikov says in his own words:

  “I was simply thunder-struck by the novelty and beauty of the Hayflick limit. I thought about this as I returned home from the University and walked along the quiet Moscow streets that were paved with gold-colored leaves on that early evening in late Fall as I made my way to the subway station13.”  

What then occurred is a rare example of unexplained insight. As he waited at the subway station he heard the roar of an oncoming train. He then had a nearly clairvoyant insight into the mechanism of mortality and immortality. As the train was coming out of a tunnel, he thought of the fact that people don’t enter the very end of the train. They enter a door near the end. Perhaps, he thought, in an analogous manner, the molecules that attach to the DNA thread to “photocopy” it every time a cell is replicated cannot copy the very end of the DNA strands, specifically the telomeres. The result would be that the cell would be just fine for some period of time, but the progressive shortening of the chromosome ends over time would eventually cause the cell to have severe damage leading to cell aging.

But then, how do we explain the immortality of the germ line and cancer cells? In the case of the immortal germ line, Olovnikov proposed that a special type of DNA copying machinery existed that had yet to be discovered, that could replicate the very ends. In these cells, the DNA could be faithfully replicated every time a cell divides, allowing the cells to live potentially forever. He published his theory in 1971 in the Russian language. He later remarked:

  “Hayflick’s finding was for me like Ariadne’s thread was for Theseus, who followed it to escape from the labyrinth14.”  

Olovnikov’s ideas were so imaginative and there were so few facts to support them that few scientists took him seriously. But some of us in Sam Goldstein’s lab found his theory attractive. First, it fit into the types of models we were thinking about; namely repeated DNA sequences as the clocking mechanism. The telomeres were thought to be rich in their own type of repeats. Second, unlike the Eco RI and Inter-Alu repeat loss models, Alexey’s model could be directly linked with cell division since he had pointed out a logical cause of the chromosome end repeat loss. Indeed, just after Alexey’s paper came out, Jim Watson, the co-discoverer of the structure of DNA had published a paper on the potential problem in replicating the ends of linear DNA molecules15. Watson observed that one end strand of DNA, called the “lagging strand” had no known means of being replicated. This came to be known as the “end replication problem.” So, this end replication problem was the first reasonable explanation for the “clock” of cell aging. Later, others were to implicate the problem lay in the other DNA strand. Regardless, we now had a reasonable mechanism that could count and remember how many times a cell has divided and “remember” the number. The number was theoretically “written” in the length of the terminal ends of the DNA. The clock would operate the way a fuse works as a clock. That is, the longer the fuse, or the longer the telomere, the longer a cell could divide before it grew old. Parenthetically, Dr. Watson served on the Scientific Advisory Board of Geron in the early days. When asked whether he knew of Olovnikov’s theoretical paper published in Russian a year before his own, he answer was, “Well, I have had at least two good ideas in my life, and the end replication problem was one of them.”

A scientific test of Olovnikov’s clock would take some time. When Olovnikov proposed his theory, no one knew the structure of the ends of DNA. Without knowing the actual DNA sequence of the ends, no one could think of a way to measure any changes in their length.

The first break came in 1986 quite by accident. Not intending to be testing Olovnikov’s theory, a Scottish researcher named Howard Cooke, was studying a segment of DNA very near the end of one of the human DNA strands. He saw that when he chopped up DNA in precise pieces, the end piece, that included the telomere, always came out in a vast range of sizes. To figure out why this was happening, he decided to look in numerous cells in the body and as a function of age. Interestingly, he saw that the length of the ends of DNA were getting shorter with age in human beings. As he stated in his 1986 paper:

  “One possible mechanism for this loss could be that somatic cells, but not the germ line, are deficient in the type of terminal transferase activity demonstrated in tetrahymena, resulting in a loss of DNA at each cell division. This could imply that the number of terminal repeats would limit the number of all divisions possible1616.”  

  A figure from Howard Cooke’s 1986 paper summarizing the first direct evidence in support of the Olovnikov’s telomere hypothesis.  

Dr. Cooke’s data supported the Olovnikov theory, but didn’t prove it. More data was needed, and a major break came when the structure of the ends of DNA was finally discovered. It was found that the ends of DNA were “capped” with an unusual repeated sequence of DNA. Like shoelaces that end with a small piece of plastic called “aglets”, so the ends of DNA are capped with the repeated sequence “TTAGGG” repeated over and over again.

Why are the ends of DNA capped with this sequence? The sequences likely result from a very ancient life strategy, but were retained by cell as a means of tagging the ends to signal a normal end as opposed to an end resulting from a broken strand of DNA. A broken strand would differ in that it would be an end without the repeats. A cell that has such telomeric repeat-less ends, can thus be identified as broken to signal DNA repair.

  The Telomere. The DNA in human cells is present in 46 pieces called chromosomes. Each end is capped with the telomeric repeat sequence that is made to specifically glow bright green in this photograph. (Courtesy of Peter Landsdorp).  

Telomere loss with Cell Aging

Once the sequence of the telomeric DNA became known, it was possible to more directly measure telomere shortening in aging cells. The way this is commonly done is that proteins that slice up DNA (but not telomeric repeat DNA) are used to digest DNA into small pieces. The DNA is then pushed through a gel to separate the DNA by size. The millions of small fragments that have the TTAGGG sequence (called Terminal Restriction Fragments (TRFs) are then visualized. As shown below, the germ line starts at about 15,000 of the DNA nucleotide building blocks. Cells enter the Hayflick limit when the average TRF length shortens to typically around 5-7,000 of the building blocks. SV40 or other tumor viruses extend cell lifespan by allowing cells to proliferate to even shorter lengths. Out of the resulting chaos (including chromosome pairs each without telomere repeats fusing as if to heal broken ones), the telomerase gene rarely gets turned on, resulting in typically a stabilization of telomeres at a relatively short length.

  The Test of Olovnikov’s Theory by Measuring Telomere Length. A. The Terminal Restriction Fragment (TRF) length of cells or various ages were measured. Cells begin life with a TRF length of about 15 thousand base pairs (kbp) of DNA and shorten to an average length of about 5 kbp at the Hayflick Limit, also known as Mortality 1 (M1). Viruses like SV40 can extend cell lifespan to an even shorter length and then the cells arrest in crisis (Mortality 2 (M2)). B. Telomere lengths of fibroblasts when they are young and then different ages through to senescence.  

It immediately became clear to many of the researchers studying the aging of cells, that the loss of telomeric DNA may offer a mechanism for why cells eventually stop dividing. As described above, a broken DNA strand would be expected to put the brakes, so to speak, on the cell’s ability to proliferate. These brakes, in turn were likely over-ridden by tumor viruses such as SV40.

Dr. Karen Prowse, a scientist at Geron in the early days was studying telomeres during the aging and immortalization of fibroblasts from a wild type strain of mice known as Mus spretus. Below is an image from one the Geron slides from those days showing the striking loss and then sudden restoration of telomere length at a time corresponding with the spontaneous immortalization of the cells.

  A Southern blot of DNA taken from fibroblasts of Mus spretus during the aging of the cells and then after the cells spontaneously regained rapid proliferation (immortalization). The DNA was hybridized to a radiolabelled probe of mammalian telomere sequences. The blot showed evidence restored telomere length at a mean population doubling (MPD) corresponding to where the cells showed a restoration of rapid proliferation and telomerase activity.  

Though such results were mere correlations in those days, and our critics (including those in the telomere community) doubted the causal connection of telomere shortening and telomerase in cell aging and immortalization, the sheer elegance of this image reinforced my conviction we were on the right track. And as we will discuss later, the search to generate another image of what I suspected would be a natural immortality and lack of telomere shortening of embryonic stem cells from Mus spretus, and then the aging and telomere shortening in mortal somatic cell derivatives, would unwittingly lead to the first successful isolation of the human counterpart: human embryonic stem cells.

Telomerase and Cancer

But had cancer cells indeed immortalized because of an activation of telomerase? In 1989 Gregg Morin of the University of California at Davis was successful in measuring the telomere-extending activity of telomerase in the immortal cancer cells known as HeLa17. While telomerase was not observed in normal mortal cells, the definitive answer on the possible association of cancer and telomerase awaited a new and sensitive means of detecting the activity of the molecule. A new and more sensitive technique we called “TRAP” allowed us for the first time to take a careful look.

A large scale study of many cancer types showed that 98/100 immortal cancer cell lines were positive for telomerase activity, while none of 22 mortal cell cultures showed telomerase present. Interestingly, 90/101 tumor samples also showed telomerase activity while none of 50 normal tissues were positive18. Not only did this support the association of telomerase with immortality, it also suggested that telomerase could be an important diagnostic and therapeutic target for managing this devastating disease.

The remaining question was, could we actually find the genes for the components of telomerase and finally prove the telomere hypothesis of cell aging and immortalization? Dr. Carol Greider, when a graduate student at the University of California at Berkeley, showed that telomeric repeats in the pond water organism called Tetrahymena were made by an enzyme that required a strand of RNA that provided the information to make the correct order of the DNA repeats. For this pioneering work on telomerase, she, Elizabeth Blackburn, and Jack Szostack won a Nobel Prize. Those of us in the aging community knew that Tetrahymena was a naturally immortal single-celled animal, reflecting the ancient immortal origins of life as described by August Weismann. But, was this enzyme actually the lynchpin key to aging and immortalization?

  Telomerase is a combination of protein and RNA. The RNA binds to the telemere and codes for the correct synthesis of ‘TTAGGG’ onto the telomere end thereby extending its length.  

Our group at Geron finally isolated the RNA component. Having this one piece allowed us to perform some critical tests of the role of telomerase. We published in 1995 that by eliminating the RNA, HeLa cells could be forced from their immortal state back to a mortal one and stop dividing after 23-26 doublings19.

More significantly, after nearly $30 million of research, we finally reported on the identification of the protein catalytic component in 199720. We knew we had the gene when we examined the levels of expression of the gene in normal mortal cells, normal human germ line cells (testis), and then cancer cell lines. The gene was always expressed only in the immortal cells.

  The protein component of telomerase when cloned showed the expected pattern of expression only in immortal cells20.  

The Experiment of a Lifetime

The experiment was terribly simple in design. The telomerase gene would simply need to be transferred to mortal cells. We would then grow normal cells to old age with and without the added gene. Of course, the DNA containing the gene had to be engineered so that the gene could not be turned off when in the cell. If the addition of the gene extended cell lifespan or even immortalized human cells, we would know that the experiment was a success.

First off, scientific research is generally a very laborious process and most experiments simply don’t work. So, we were very much prepared that the experiment would not work first time around, or perhaps ever.

As it happened, Len Hayflick was visiting Geron around that time and the film crew interviewing him asked him for a graphic illustration of how cells are removed from the body and placed in culture. In his typical robust style, Len borrowed a sterile scalpel from our lab and with the cameras running, sliced a piece of skin from his shin while the cameras were running.

After the filming, his walked into my office, skin in hand and asked whether I needed a skin donor. I put his skin in culture as shown below.

  Len Hayflick’s explant and the first immortality experiment. The piece of Len’s skin is in shadow on left. Skin cells can be seen emerging from the tissue onto the culture flask in vitro on the right.  

My real interest, however, was more of an historic one. I have always had an interest in the history of science. When I first arranged financing for the telomere project for Geron from the venture capital firm Kleiner Perkins Caulfield and Byers in 1992, I bought Len’s old lab equipment to set up our first lab. Now, some five years later, I hoped to use his cells in the first test of telomerase in human cells.

In the weeks that followed, I plotted the aging of Len’s cells along with the cells into which the newly-cloned gene had been introduced. With every passage of the cells, the results became more and more clear. The cells with the added telomerase gene were living longer and longer. In fact, they showed no evidence of aging at all.

To properly test the result however, we needed more experiments a. nd a much more carefully-designed study. We had to be sure of the result if we were to publish the result as a fact. In collaboration with Woody Wright and Jerry Shay at the University of Texas Southwestern Medical Center at Dallas, Geron’s team published the first result in 1998. One gene was sufficient to stop the aging of human cells21.

I had the pleasure of having dinner with Len around that time. He suggested dinner at a restaurant overlooking the Pacific Ocean. Over dinner I updated him on the experiments. Len turned and looked out over the ocean and the setting sun. “It just can’t be that simple…” he said out loud. Remember, the data for years argued that there were over a hundred documented changes in aging cells. But our data suggested that there was only one gene ultimately in control of the process. For some reason, that gene (the telomerase catalytic component) was turned off in somatic cells, leading to telomere shortening as the cells divided, while staying on in the germ line cells allowing babies to always be born young. The next question was whether we could use the gene to test the role of cell aging in human aging itself.

The Evolution of Aging and Future Prospects

There has been a lot of speculation on why we evolved as a mortal species. A simple answer, but by no means the certain one, is that to live as long as we do as humans, we needed to put in place safeguards to prevent cancer. After all, mice often get cancer and die in just 2-3 years of age. So, one possibility is that by turning off the telomerase gene in the body, we could live long enough to parent children and limit cancer, without causing cell aging. Now that people live much longer than necessary to simply have children, we see the deleterious side of somatic cell aging. We see a surge of age-related degenerative disease linked to the aging of cells in tissues throughout the body.

So, is it possible to turn telomerase back on in the human body to test the role of cell aging in human aging, and to possibly reverse or prevent age-related degenerative disease? There are weak activators of the gene that have been identified. Though, to my knowledge, they are very weak and have minimal if any effect on telomere length in the body.

Are such compounds safe? Since the activation of telomerase correlates with cancer, there certainly are justifiable concerns that such therapy could cause cancer. My best judgment, however, is that older individuals at risk of serious disease are probably safe to try extracts based, for instance, on the astragalus root. However, otherwise healthy people taking these extracts for long periods of time, might, in my opinion, be putting themselves at increased risk of cancer for minimal health benefits.

In the case of life-threatening age-related disease, is there a more potent method of resetting cell lifespan in the body for therapeutic effect? One method of accomplishing this could be that of gene therapy. As was done in the first laboratory tests of telomerase, we could place the gene in viruses, and use them to spread the gene throughout the body, adding the gene into cells, and potentially resetting the telomere clock.

Gene therapy has been very slow to advance as a therapeutic strategy. Nevertheless, in the case of cells that can be removed from the body, treated with such telomerase gene therapy treatments, and then re-introduced into the body, might show promise. Other strategies based on stem cell technology will be discussed next.

  Telomerase gene therapy. It may be possible to package up the gene for the catalytic component of human telomerase into a virus and use this as a means of gene therapy, resetting the telomere clock of cell aging in cells and tissues of the human body.  

  References

1. Goldstein, S. 1969. Lifespan of cells in progeria, Lancet, 1(7591): 424.
2. West, M.D. 2003. The Immortal Cell, Random House, New York.
3. Hayflick, L. 1980. Cell Aging. In C. Eisdorfer (ed.) Annual Review of Gerontology and Geriatrics, Vol. 1, Springer Publishing Co. New York.
4. Hayflick, L. 1990. Unscrambling an egg. Science 248: 1281.
5. Williams, G.C. 1957. Pleiotropy, Natural Selection, and the Evolution of Senescence, Evolution. 11: 398-411.
6. Scherer, W.F., Syverton, J.T., and Gey, G.O. 1953. Studies on the propagation in vitro of polioviruses. IV. Viral multiplication in a stable strain of human malignant epithelial cells (strain HeLa) derived from an epidermoid carcinoma of the cervix. J. Exp. Med. 97(5): 695-710.
7. Skloot, R. 2010. The Immortal Life of Henrietta Lacks. Random House, New York.
8. Girardi, A.J., Jensen, F.C. and Koprowski, H. 1965. SV40-induced transformation of human diploid cells: Crisis and recovery. J. Cell. And Comp. Physiol. 65: 69-84.
9. Ohnuki, Y., Lechner, J.F., Bates, S.E., Jones, L.W., and Kaighn, M.E. 1982. Chromosomal instability of SV40-transformed human prostatic epithelial cell lines. Cytogenet. Cell Genet. 33: 170-178.
10. Dell’Orco, R.T., Mertens, J.G., and Kruse, P.F., Jr. 1973. Doubling potential, calendar time, and senescence of human diploid cells in culture. Exp. Cell Res. 77: 356-360.
11. Shmookler Reis, R.J. and Goldstein, S. 1980. Loss of reiterated DNA sequences during serial passage of human diploid fibroblasts. Cell 21:739-749.
12. Shmookler Reis, R.J. Lumpkin, C.K. Jr., McGill, J.R., Riabowal, K.T., and Goldstein, S. 1983. Extrachromosomal circular copies of an ‘inter-Alu’ unstable sequence in human DNA are amplified during in vitro and in vivo ageing. Nature, 301: 394-398.
13. Olovnikov, A.M. 1996. Telomeres, Telomerase, and Aging: Origin of the Theory. Exp. Gerontol. 31: 443-448.
14. Olovnikov, A. M. 1971. Principles of marginotomy in template synthesis of polynucleotides. Doklady Akad. Nauk SSSR, 201, 1496-1499.
15. Watson, J.D. 1972. Origin of concatemeric T7 DNA. Nat. New Biol. 239: 197-201.
16. Cooke, H.J. & Smith, B.A. 1986. Variability at the telomeres of the human X/Y pseudoautosomal region. Cold Spring Harbor Symposia on Quantitative Biology 51:213-219.
17. Morin, G.B. 1989. The Human Telomere Terminal Transferase Enzyme is a Ribonucleoprotein that Synthesizes TTAGGG Repeats. Cell 59:521-529.
18. Kim, N.W., Mieczyslaw, A.P., Prowse, K.R., Harley, C.B., West, M.D., Ho, P.L.C., Coviello, G.M., Wright, W.E., Weinrich, S.L., and Shay, J.W. 1994. Specific association of human telomerase activity with immortal cells and cancer. Science, 266: 2011-2015.

19. Feng, J., Funk, W.D., Wang, S-S, Weinrich, S.L., Avilion, A.A., Chiu, C-P., Adams, R., Chang, E., Allsopp, R.C., Siyuan Le, J-Y., West, M.D., Harley, C.B., Andrews, W.H., Greider, C.W., Villeponteau, B.V. 1995. The RNA Component of Human Telomerase. Science. 269: 1236-1241.
20. Nakamura, T.M., Morin, G.B., Chapman, K.B., Weinruch, S.L., Andrews, W.H., Lingner, J., Harley, C.B., and Cech, T.R. 1997. Telomerase catalytic subunit homologs from fission yeast and human. Science, 277: 955-959.
21. Bodnar, A.G., Ouellette, M., Frolkis, M., Holt, S.E., Chiu, C.P., Morin, G.B., Harley, C.B., Shay, J.W., Lichtsteiner, S., and Wright, W.E. 1998. Extension of life-span by introduction of telomerase into normal human cells. Science 279: 349-352.

 

© Copyright 2014 Michael D. West, All Rights Reserved

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