Disease Models & Mechanisms 2, 534-537 (2009) doi:10.1242/dmm.003418 A MODEL FOR LIFE Published by The Company of Biologists 2009

Telomeres and Tetrahymena: an interview with Elizabeth Blackburn

Elizabeth Blackburn knows that loose ends contribute to aging and many of its associated diseases. She, together with Carol Greider and Jack Szostak, received the 2009 Nobel Prize in Physiology or Medicine for their work on the synthesis and function of telomeres, the unusual DNA sequences at the ends of chromosomes. Telomere changes are now recognized in human diseases ranging from cancer and cardiovascular disease to depression. Here, she discusses her approach to mentorship, how scientists might inform public policy, and new directions in telomere research.

elomeres are made of repeating that allows you to work efficiently. This phi- DNA sequences at the end of losophy was especially important at this chromosomes that enhance their time, back in the seventies, when there were stability and protect their ends not many DNA technologies available. Joe during replication. Changes in found that Tetrahymena have high copy

DMM telomereT length, usually a shortening of the numbers – literally tens of thousands – of sequences, are associated with disease. tiny, short linear chromosomes. My PhD As we age, telomeres become shorter. training in Fred Sanger’s lab was in the Elizabeth Blackburn and Carol Greider dis- world of DNA sequencing, which was still covered the enzyme, telomerase, that just beginning. The so-called ‘Sanger makes and can replenish the telomeric se- method’ for DNA sequencing was still quences at the ends of DNA. These initial being invented, so I was using techniques of discoveries used simple, single-celled or- radiolabeling and piecing DNA sequences ganisms and lots of radiolabeling. As a together. You had to sequence DNA from postdoc in Joe Gall’s laboratory, Elizabeth the very ends. I realized that, for big mole- Blackburn kept her eyes fixed on the pear- cules, there was very little access to most of shaped protozoan Tetrahymena, and in her the sequence. Remember, this happened in hands, this single-celled ciliate soon laid the precloning days, which is now hard to foundation for telomerase biology and a imagine! You had to have short abundant Elizabeth Blackburn © 2007 Micheline Pelletier. better understanding of chromosome molecules because of the problem with structure, aging and disease. background noise. I decided on in the 1970s that some ciliates had DNA in Disease Models & Mechanisms Tetrahymena because it had abundant short the form of short linear molecules that were What lead you to focus on such a simple linear molecules and I thought I could use amenable to direct DNA sequencing by end- organism as Tetrahymena to understand the available sequencing techniques to see labeling techniques. The right molecules in chromosome organization? what was really at the chromosome ends. yeast were not known until we used a special I used Tetrahymena because it is a system Joe Gall was a great mentor and model. trick by adding Tetrahymena telomeres to that is good for doing biochemistry that re- Having a good mentor yourself gives you a the end of a linear yeast episome, which quires large amounts of biological material, fantastic jump-start on trying to become a allowed for molecular cloning of yeast telom- and because it contained a very large number mentor. Once you’ve experienced working eres. This yeast work came later. It was useful of short chromosomes that are linear. with a good mentor, you realize just how and yeast are small inexpensive organisms My postdoctoral adviser, Joe Gall, had a valuable good mentorship can be. that you can grow easily and do lots of ge- very good sense that important biological netics on fast. There were many powerful processes are conserved throughout evolu- The current literature reflects a sort of genetic tools for the yeast system, but the tion. So, you choose a model organism that alternation between the use of ciliates Tetrahymena system had the biochemical you can most readily get your hands on and and budding yeast as models to under- advantages. So, the telomere and telomerase stand telomeres and their function. Is work began in the ciliates. one model better than the other? Elizabeth Blackburn is the Morris Herzstein Professor People were using budding yeast as a How might telomerase be used as a ther- of Biology and Physiology at the Department of common model when the initial work was apeutic target in people? Biochemistry and Biophysics, University of California, San Francisco done, but at first it was not good for under- I think that it is helpful to think of telomeres e-mail: [email protected] standing telomerase. David Prescott showed in two different contexts. In cancer cells,

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changes in telomerase promote certain UCSF colleagues. We think this is a good telomerases are doing other interesting cancer characteristics and make some cells approach but at the moment it involves a things. They have a night job as well as a day immortal. Almost immediately, people re- gene insertion, which may or may not be a job. alized that cancer cells had telomerase. In good thing to do. It is just a little telomerase It is still not clear what is going on, but 1989, Greg Morin reported the first human RNA gene that is inserted, with 451 nu- the cell program is changing. So, ideas for telomerase isolated from HeLa cells, using cleotides in the coding sequence. It is not a polymerization-independent functions of the same methods that we had used in big gene and we have collaborators who use telomerase are starting to emerge. Steven Tetrahymena. Others did much broader clever targeting methodologies, such as im- Artandi at Stanford has evidence that the surveys later in the 1990s and showed that munoliposomes that target overexpressed telomerase protein affects Wnt signaling. lots of telomerase was present in cancer receptors on cancer cells. All of this is pro- That would not be expected if the role of the cells relative to less telomerase in normal gressing very slowly, but teaching us quickly telomerase in cancer depended on telomere cells. The simple question was, ‘if the that all cancer cells really hate having mu- length alone. Telomerase may affect im- cancer cells are made immortal by telom- tations added to their telomeres. portant cancer stem cell-like properties in erase, could you turn the telomerase off?’ There is another exciting context in cells. This is exactly the kind of thing you This idea could be tested by inhibition of which to think of telomerase. Experts in the really want to target therapeutically, and the telomerase. There are now some early field had a totally good rationale of why results are quite unexpected. Phase I experiments looking at inhibitors of telomerase activity would be increased in telomerase. I think that cancer cells. Cancer If it was possible to manipulate telom- the search for inhibitors Telomerase may affect cells, of course, are very erase, either its activity or level in cancer, of telomerase is one that important cancer stem abnormal in lots of ways how does the evidence that telomerase can be addressed best by and most of their abnor- has a fundamental role in the cell pharmaceutical compa- cell-like properties in cells. malities contribute to program and ubiquitous signaling path-

DMM nies. This is exactly the kind of their immortality. We ways affect its therapeutic potential? What intrigued me, thing you really want to did a very simple exper- That I don’t know, because a cancer cell is was really trying to un- iment to determine the a whole different beast from a normal cell. derstand what telom- target therapeutically, and influence of telomerase A cancer cell doesn’t have proper check- erase was doing in the results are quite level in cancer cells. In points, but instead has all sorts of anti- cancer. Our experiments unexpected my lab, Shang Li apoptotic pathways activated. Its signaling in Tetrahymena proved knocked down the is really different from a normal cell. We that telomerase is a ribonucleoprotein telomerase RNA with standard RNA inter- don’t want to knock telomerase down in reverse transcriptase. If we mutated the ference. We expected to have to sit around normal cells because maintaining telomere template of a cloned telomerase RNA se- and wait for a long time before the telom- lengths is probably a healthy thing. So, if quence, we found that the mutant repeats eres ran down and the cells stopped divid- you’re going to inhibit telomerase or knock were inserted into the telomeres. As soon as ing, but to our great surprise, the cells re- it down in cancer cells, you want to be you add these mutant repeats to the telom- sponded very quickly. They didn’t immedi- pretty careful about specificity. Based on in eres in Tetrahymena cells, the cells look ately stop dividing but they immediately vitro data from our lab and others, I’m con- very sick. So in the mid-1990s, I was talking slowed. We looked at their gene expression vinced that any cancer therapy that in- with cancer researchers at UCSF profiles by microarray and found a changed volved telomerase as a target would be a Disease Models & Mechanisms (University California, San Francisco) and profile that appears quite quickly after combination therapy. Telomerase would said, ‘I‘ve got this interesting finding that telomerase knockdown. not be the only target because it may be suggests that we could add mutant se- Since we were changing telomerase RNA dangerous to knock telomerase all the way quences to telomeres to kill cancer cells by knocking it down, it suggests that there down in normal cells. right away’, but they were initially much is something special about reducing telom- In normal cells, telomerase creates a more interested in telomerase inhibition. erase expression which is different from in- whole different picture because these cells We did some early screens but never pub- hibiting the enzyme and leaving the ri- have highly regulated telomerases and lished them. We were not, ourselves, bonucleoprotein concentration high. The telomere lengths. Normal cells show experts in developing small molecules. experiment that we did was in melanoma changes in telomeres as they age. In 2001, My idea was to use the telomerase to and when we just turned the telomerase ac- Inderjeet Dokal reported initial genetic make cancer cells really sick. It seems ef- tivity down a bit, the cells continued to findings that mutations in the RNA com- fective to use the naturally occurring high grow, just a little more slowly. Then we ponent of telomerase cause a severe type of concentration of something that is already knocked down the level of the telomerase premature aging. It’s very clear that these in cancer cells to turn against the cancer cell RNA component so that the total telom- mutations were in the telomerase RNA and from within. We are now at a very, very erase ribonucleoprotein concentration was now many more have been identified. We early preclinical stage with this idea. We are lower. When we did that, the melanoma looked at the in vitro telomerase activity of able to induce a very robust apoptotic re- cells became more differentiated and less the mutants that occur in human disease. sponse. We are testing the idea in mouse malignant. It is a fast-acting effect. The cells We took some of the mutations that Dokal models, using delivery methods with clever do not have time to run their telomeres described and showed that they, in effect, liposome-derived nanoparticles, with our down and then go into crisis. This hints that knock telomerase telomere synthesis activ-

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ity out – either completely, or down to use to analyze people. We have even used erase is a cellular ribonucleoprotein so you about 1%. People with these mutations have cognitive dietary restraint, which is also need to have actual cells. It is a mutual ed- one good gene and one bad gene. In these striking. It’s basically a fancy name for ucation. There was so much that I did not situations, people get terrible bone marrow people who are under stress and have eating know about clinical design, so I always work failure and other symptoms like fibrosis. It disorders; they are constantly trying to diet with collaborators. They know what makes is clear that defective telomerase RNA but they don’t actually lose weight and they a good clinical study and whether a study results in defective telomerase activity, beat themselves up about it. It’s a common should take into account certain things – which in turn is associated with diseases in- problem in our very food-exposed world. for example, who is on medications – to be volving bone marrow failure and even pul- There are various questionnaires and scales able to draw important conclusions. They monary fibrosis. So, common conditions that are validated to measure this. Sure know the pitfalls that I’m now learning. It’s are caused by telomere dysfunction. enough, when we look at telomere length in very interesting because we really have to Curiously, telomeres seem to be limiting for people, there is an inverse relationship talk and have lots of face-to-face meetings humans and there was no a priori reason to between telomere length and pessimism, to make sure that we’re getting each other’s expect this result. and between telomere length and eating language. disorders. This doesn’t demonstrate causal- It seems that changes to telomeres may ity, but it indicates a strong association that You have a reputation of being a good influence many areas of human disease. is quite striking. mentor. What qualities do you think are From about the year 2000 onwards, an im- necessary to be an effective mentor? pressive compendium has accumulated that Does stress cause telomere shortening? I try to remember what it is like for is derived from all sorts of cohort studies Most of these big cohort studies weren’t someone at the early stages of their career. around the world, showing that telomere looking at stress directly, but it is a really in- Sometimes I draw on my own personal ex- shortness is associated with just about all teresting relationship. We became involved perience and sometimes it’s what I’ve

DMM the major diseases of aging. Right now it is in the stress part through a colleague of learned from other people. I do talk a lot all association, but very interesting. mine at UCSF, Elissa Epel, who was working with younger colleagues. It’s important to Scientifically, one has to be very careful not initially with Nancy Adler. They both look just remember what it was like, although it’s to say anything about causality except for at physiological effects but are really inter- really easy to forget. I remember positive the rare genetic diseases. But, over and over ested in the world of psychology. Elissa e- things about my postdoc mentor, Joe Gall. again, telomere changes are associated with mailed me in 2000 and said could she come When Joe said positive reinforcing things to everything from cardiovascular disease, and talk to me about her ideas to look at me, I remember how much it meant and death from cardiovascular disease, risks of chronic stress. She wondered, ‘Has anybody how very insecure I was. I was insecure cardiovascular disease, diabetes, diabetes ever looked at telomere maintenance in enough that these things were important risks such as insulin resistance, vascular de- people with chronic stress?’ and I said, ‘No, for me to hear. For him to say ‘you are a mentia, to osteoarthritis. The list goes on but we should look’. She gathered her good scientist’ told me things that I didn’t and on and the correlation is always in the cohorts very, very carefully. Richard allow myself to think. same direction: shorter telomere length is Cawthorn in Utah assayed the telomere Scientists often forget something that associated with more disease. The associa- lengths and we analyzed the telomerase ac- most of the world realizes, that you actually tion is absolutely solid now because it has tivity. We did all of our samples blinded. have to say positive things to people too. I been found in so many cohorts that it Elissa called up very excited and said, ‘Wow! try to remember the experience I had, but Disease Models & Mechanisms cannot be a statistical accident. What does There is really a correlation’. The more I have to be reminded sometimes because this mean? That’s a really exciting question severe the stress that a patient reported, the in science we are trained to be critical. We and I don’t know yet. I am skeptical that the more the telomerase activity was damped just go straight, very efficiently, to the faults answer is going to be very simple. down. This opened the door for us to look and often we forget that it’s quite difficult to at so many other things. So, it seems that be on the receiving end of criticism, even Are there models to support the correla- the best model for human telomeres is when the intention is good. So, I try to put tion between short telomeres and humans, and that’s our model system now. myself in other people’s shoes. Often when disease? We talk a lot with our clinical colleagues people are cocky on the outside they’re not In mouse models, only when telomerase is because there are really two different cul- on the inside. Rather, they are really very completely ablated is there a phenotype, tures between scientists and clinicians. You vulnerable and I try to stay aware of that all which is generally delayed. Mice normally have to learn to talk their language and they the time. die naturally at 2 years of age and still have have to learn enough about telomeres and long telomeres. But humans live 80 years, telomerase to see what makes sense for Does being a good mentor have any which is a very different life expectancy their patients. One of our colleagues once direct influence on your career? from mice. I’m very intrigued by what had a beautiful study and he sent us samples If people do come and ask me to be a telomeres do in humans. To address it, we to test. My postdoc looked at them and said, mentor, it is much the same as when people collaborate with people who do clinical ‘Oh, where are the cells!?’ and he said, ‘Oh! come and talk to me about other things. studies. I thought you wanted serum’. We realized The time commitment is minor and doesn’t We have found that pessimism is one of that we hadn’t talked carefully enough take time away negatively from something the psychometric measures that one can about what we were looking at, that telom- precious. The positive thing is that men-

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toring allows me to have really good stu- of life there is much more circumspection. But I wouldn’t have liked to enter this dents and postdocs. People are now smart Our culture is direct. If we think it’s the area when I started in the 1970s. At that enough to take someone’s mentoring rep- case, we say it clearly and this approach is time, molecular biology was all the rage and utation into account when choosing a lab. valued. So, it seems to me that scientists can that was where things were exciting and I was told that Joe Gall was a terrific mentor offer public policy a different way of ap- fast. I don’t think that neuroscience had the when I went to Yale. That was a really proaching questions that is clear headed, capabilities then that it has now. helpful thing for me to know. When you focused on evidence and that clearly dis- have really terrific people in your lab, it cusses the issues. I keep saying that we’ve Have you looked at telomeres in the brain helps the science so much. got to get the science right, because that is or neurons? I have never really thought of myself as what we do as scientists. I used to joke that, No, I’m more interested in how the brain a great mentor. But, I think that being a in that respect, it is just like our lab group sends all sorts of messages to the rest of the mentor means caring about people so that meeting. So, I think the best thing a scien- body, such as vagal innervation. All we used people want to come and do good science. tist can do in public policy is to try to get the to know about the brain is that it churns the And the people who come are fantastic. scientific facts right. stomach and beats the heart. But there is This, of course, makes the science happen. even a vagal innervation that goes right to I don’t know how much of the lab So the critical directness that is charac- stem cells in the spleen. It is physically right dynamic is cause and effect. If I were a hor- teristic of scientists should be saved for there. I also learned recently about pretty rible mentor, if I were known to be really public policy and not for mentoring? direct effects of the brain on the immune mean or something like that, then would I Right! That’s a really good comparison. system, and these are just a tiny example of still attract really good scientists that were With mentorship, to be most effective, we what the brain must do. I would love to different kinds of people? We all know have to actually do what we’re not trained to know how it works. The brain integrates the people who are very poor mentors but who do. The evidence is that mentoring is more whole organism much more than we ever

DMM do really good science. The question is, ‘is successful if you do it in certain ways. That’s thought, and this is an area of emerging there a cost for their poor mentorship a kind of training too. It’s just not the science. The molecular biology has gone style?’ Maybe their science could have been subject matter that we’re as used to. very far and now we have to re-integrate all even better. of this information about the human or- If you were to start out all over again as ganism. I think the human is the best one to After a frustrating term as an invited a new scientist, is there a different field study for this. member of President Bush’s Council on where you would choose to work? Bioethics, do you have any advice about If I had to start again now I would choose That will make it a difficult area to how scientists might inform public neuroscience, since it has advanced so model. policy in a useful way? much recently. I see how important this Yes, that’s what the great molecular biolo- Yes, I really do. I went on the Bioethics area is from the chronic stress tests where gist, Sydney Brenner said: ‘The model for Council wondering what I could bring to it. we can see how the brain feeds in to all of human organisms is humans’. I think there I know how science works, I know good the rest of human physiology. That’s what are some very powerful mouse models and science from bad science and I know the I would love to have as my field. animal models that have been very power- field. I’m not directly a stem cell scientist, I’d like to understand how the brain com- ful for highly conserved things. There’s no which I knew would be a big question for mands the body because in the field of question that they are very important. Disease Models & Mechanisms the Bioethics Council, but I do know cell telomeres we see the role that it plays. It is We greatly appreciate Elizabeth biology and I’m in the general field, so I also a very interdisciplinary field, bringing Blackburn’s retelling of her exciting story could speak about it. I also felt that I could so many different physiological conse- and her thoughts about mentorship, politics think about evidence, since that is what I am quences together. Once the view of medical and the future of science. Kristin H. Kain, trained to do. That’s what I think scientists science was that the impact of the brain on Associate Reviews Editor for DMM, inter- really can bring to public policy, we are very physiological disease was complicating. viewed Elizabeth Blackburn. This piece was good at listening to arguments and we can Separate cultures and ideologies formed edited and condensed with approval from tell when an argument is weak. We are and I think there was a sort of dismissal of the interviewee. trained to think critically and I think that’s brain effects as just psychosomatic. Wait a DMM congratulates Elizabeth very useful for public policy. minute, that’s the whole point! There are Blackburn, Carol Greider and Jack Szostak Scientists have a culture, I discovered, of real effects of the brain on disease and the for being awarded this year’s Nobel Prize in being rather honest. We tend to just say science for understanding this was not ade- recognition of their exciting telomere re- things, whereas I think in many other walks quate for a long time. search.

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