Aging and disease

Elizabeth Blackburn during an interaction with students at the National Centre for Biological Sciences, Bangalore.-K. MURALI KUMAR

PROFESSOR Elizabeth H. Blackburn is a Tasmania-born American scientist whom Time magazine named in 2007 as one of the 100 most influential people in the world. Widely seen as a potential candidate for the Nobel Prize, her ground-breaking work concerns the ends of eukaryotic chromosomes, called telomeres, which serve as protective caps for the genetic information in the cells, similar to, as she likes to describe it, the end caps of shoelaces, which are called aglets.

She is also credited with the discovery of the cell enzyme telomerase, which replenishes the telomere. Research over the years in the area has led her to demonstrate the significant role that telomeres and telomerase have in human health, in particular age-related diseases such as cardiovascular disease and cancer.

Sixty-year-old Elizabeth Blackburn is at present the Morris Herzstein Endowed Professor of Biology and Physiology at the University of California, San Francisco (UCSF). After graduation from Melbourne University in 1972, she earned her Ph.D. from the University of Cambridge under Fred Sanger. In 1978, she joined the faculty of the University of California at Berkeley and in 1990 moved to the UCSF.

Elizabeth Blackburn was in India in February on a lecture tour as the featured speaker of the 2009 Cell-Press-TnQ India Distinguished Lecture Series, which included talks on Chromosome Ends and Human Health and Disease in Bangalore, Hyderabad and New Delhi. The lecture series has been instituted to bring internationally renowned scientists face to face with the scientific community and the public in India by organising scientific talks in select cities and institutions each year.

Elizabeth Blackburn gave Frontline a wide-ranging interview, first in Bangalore and then in New Delhi, on her scientific work. Excerpts:

Your work is concerned with the role of chromosomes in human health and disease. What aspect or component of a chromosome is critically involved in this?

The part that we work on is the very ends of chromosomes, which are called telomeres. Much like the tips of shoelaces, telomeres are molecular caps that protect chromosomes [which carry the genetic information inside the nucleus of a cell]. Because without special protection the chromosome ends are susceptible to being chewed away and frayed away by natural processes taking place inside cells. So we focus on how those ends carry out their biological roles and what they consist of in terms of molecular features and how an enzyme that we discovered some years ago, called telomerase, replenishes the ends as they wear out.

Telomeres wear down with time, and when these become so worn that they cannot sustain cell division any more without causing chromosome instability, cell division stops [senescence]. This is what leads to aging. One characteristic aspect of aging is the increased susceptibility to disease, particularly age-related diseases such as cardiovascular diseases and cancer. Then the question is: Over time can the balance of telomeres wearing down versus the telomeres being replenished [by the action of telomerase], first of all, affect human disease? And the answer is yes. Then, is there anything that we can learn which changes the balance between the wearing down and the replenishment that might have impact on human diseases?

You chose Tetrahymena thermophila to study telomeres and telomerase and you also remarked in your lecture that the organism was a great experimental system. Would you elaborate?

I had been in Cambridge, and Fred Sanger and I were working on independent methods of sequencing DNA [deoxyribonucleic acid]. I realised that one of the entry points to get to that is the ends. This was before DNA sequencing was working at all. There were only ways of patching together information. I realised that the ends are something that I could do. And nobody knew what the ends of chromosomes were like. So those things came together.

When I went to Yale, I applied to work in the lab of Jo[seph] Gall, who discovered that the organism had small linear chromosomes. He had shown that in the nucleus of its cells were very, very tiny chromosomes. They were of a particular kind. But there were 10,000 to 20,000 copies in the nucleus, and they were all very small, which meant lots of ends. He pointed out, Look, these are good organisms because you can grow plenty of them just in big flasks, and they will multiply well and so you can get enough material to do direct molecular studies.

That was the practical reality of saying [that] this would actually help me molecularly understand the ends because normally chromosomes are so long that very few ends of DNA are left. Because methods of sensitivity then [were such that] you still needed enough purified material to work with. That was the reason.

Before you elucidated the telomere structure and the importance of telomerase, which you discovered, what was the general thinking like?

Well, there wasnt any. What we showed in the late 1970s was what [the] telomere DNA structure looked like. And data were perfectly clear and people accepted that. [But] the idea that there would be telomerase [was] something that hadnt been seen before. Then, internally you have to do a lot of convincing yourself that the data are not an artefact.

So before I presented it, my graduate student Carol Greider and I had already amassed information that convinced us from studying what the reaction was and doing experiments that would really rule out artefacts. We pretty much could answer in our minds that this wasnt just an artefact where, say, some random DNA polymerase was copying and contaminating fragments of DNA that came through from the cell extracts. So we had really ruled out why this might have been not a new enzyme but just some variant enzyme reaction carried out by enzymes that people already knew about.

Though a scientist who had expressed views earlier about how telomeres might be replicated based on paper models had expressed reservations about our work, I didnt take it as a serious critique. But we were really thinking: Were we being fooled? For about a year, we were just going back and forth trying to think of experiments that could undercut [the conclusion] but it was really something new.

How does this wearing down of telomeres happen?

There are a couple of known reasons as to how the telomeres wear down. We can ask a bigger question as to why but lets answer the how first. It is twofold.

One is DNA replication, which has to happen for the genetic material to be completely copied so that there are two copies of the genetic material before a cell divides and then each copy goes into the two daughter cells. That molecular machinery made of enzymes and associated proteins is very good at copying very accurately almost all the length of the entire chromosomal DNA with very high accuracy. But for some strange reason that a human cannot think why, it cannot copy the very ends of a linear DNA. Its just an odd deficiency in this enzyme that is otherwise engineered to be so exquisitely very accurate and thorough in copying.

Bacteria have circular chromosomes. Bacteria are much smarter than usthey dont have this problem. But eukaryotes everything from humans to plants to yeasts to single-celled organisms except bacteria have linear chromosomal DNA. The other reason is that there are enzymes in the cell that naturally chew away on DNA ends because of the repair processes that they sometimes can carry out. But the primary reason is the inability of the DNA replication machinery to carry out copying at these very ends. Telomerase is the enzyme that adds the extra DNA to the ends. Basically, it just adds a buffer of DNA that does not code for proteins but acts as an attraction for protective proteins, which form a kind of a sheath around the ends of the chromosomes.

Is there something like a critical length for these telomeres when susceptibility to diseases begins to develop?

We do know the average size [of telomeres] for humans. Its surprisingly large; its a few thousand nucleotides, or building blocks of DNA. Thats in normal cells in the body. Its interesting that other creatures have smaller telomeres, and just a couple of hundred nucleotides is plenty there. Why such a large amount of DNA in humans? The way proteins get attracted to the telomerated DNA to form a sheath is very dynamic; the proteins come on and off a little bit like a swarm of bees. If you looked at a swarm of bees, there is a shape; there is a protection there. But if you looked closely enough, you get to see that the individual bees that its made up of may be changing all the time, flitting in and out. You see an overall shape of protection, but its changing every minute, probably every second.

[Similarly,] it must be something to do with the rates at which proteins come on and off. When its only a hundred nucleotides, the rates at having the sheath being formed as opposed to being unformed seems to be unsatisfactory. We dont know why from a physical point of view, we just have to infer. So in humans we get a rough idea; we know that telomeres cant really fall below that length for cells being able to keep renewing themselves, keep multiplying, which is what cells have to do to keep the immune system being built up, the skin and all parts of the body that have to continue to maintain different shapes throughout life.

Its even true of brain cells, which people always thought they didnt after you became an adult and the brain cells youve got are what you are stuck with. In a rough way thats true, but now its clear that there are actually stem cells that continue to give new neurons throughout life. So many, many tissues have to keep producing new cells, and its very important for them to continue to function throughout life.

Some cells, such as skin cells, white blood cells, germ cells and stem cells, require continuous replenishment; there are others such as the brain cells that do not require as much and there are others that perhaps do not replenish at all. Are the telomeres in these of varying lengths depending upon how much replenishment is required?

It would seem like that. Even within different white blood cells we have shown for the first time that mature white blood cells have quite distinct levels of telomerase which will be a determinant of how well the telomeres are maintained and that tracks quite well with how much telomere lengths on the average that the white blood cells have. So this is quite truly well regulated even just among white blood cells.

And there is some indication from very incomplete studies that there are rough correlations in a person between say skin cell types and the white blood cells, and for different people you have different telomere lengths. And genetics does make a difference to that.

But also non-genetic factors can influence how much telomeres are replenished and how much they are worn down. So its all about a balance here. Whats curious is that humans have evolved to have very low [level of] telomerase. Is there some reason for this? Or is it just because evolution took place when the selection was not based on how many decades people would live out? People were more likely to die of disease or malnutrition or things like that and telomeres running down wasnt going to be a primary determinant of how many children they would have.

Now, of course, weve overcome many of these things, and its time for this to play out. We notice as people get older their telomeres wear down. And thats what gives us a lot of hope that we might be able to modulate that. Its not inextricable as people thought and now we [also] know its different from person to person.

When you say that diseases afflict humans because of telomere shortening, do you mean that the cells that are affected by the diseases are the ones that have shorter telomeres?

Now this is interesting. You mustnt get this impression. I will tell you about the cells that we know the most about. Those are the white blood cells, which are the immune system cells. So when you look at them, you are getting a very good picture of how the immune system is doing. And you might say if cardiovascular disease is important or cancer is important, why should we care about the immune system?

Lets take cardiovascular disease. More and more it is realised that, in fact, the immune system produces inflammatory responses, which then have effects on the heart and blood vessels. Thats quite a contributor to heart disease. And people know that inflammatory responses exacerbate and may probably be linked to heart disease. So, actually when we are looking at white blood cells, that might be very much directly related to whether its going to be cardiovascular disease. We used to look at cholesterol and things like that. In new studies they looked at blood proteins called C-reactive protein. It is a measure of the fact that theres been an inflammatory response of some kind or the other. And that turns out to be quite closely linked to cardiovascular disease.

And similarly cancer. For years its been inferred that the immune system does a valiant job for a long time in trying to contain and kill off cancer cells. Eventually, it loses its ability to do this for one reason or another and then the tumour appears. Immunologists have known for a long time that by the time someones cancer appears, the immune system is actually not the same as it was before. A very interesting study [was] done looking at whether a particular predisposition for oesophageal cancer will progress to cancer. They studied people who had this predisposition called Barretts oesophagus, where the oesophagus gets somewhat irritated. They followed these patients to see who progressed to getting actual cancer. And then they tried to understand whats the risk for that and they found that they had short blood cell telomeres.

This again is very consistent with the immune system being a good indicator [of] whether the cancer might develop even though the cancer is not the cancer of the immune system. So all these lines of evidence start converging in a way that makes you think probably we should take notice of this. As a scientist one would be cautious to say: Oh great! This is going to explain everything. Thats very tempting in biology. But theres a consistency here that is hard to ignore. Thats coming from different lines of evidence from different studies in different diseases. We keep seeing something working in the same direction.

You have found that non-genetic factors like stress are also associated with telomere shortening, and hence susceptibility to disease. Is the following picture then correct? Because of stress and environmental factors, the telomerase activity gets reduced in the immune system cells of whatever kind, thus depleting them, and this increases the susceptibility to disease

Or the stem cells that give rise to them throughout life. If the telomerase activity gets damped down in those, then that will have big consequences. In the extreme genetic disease models, we know that the immune system stem cells do deplete. Thats a very factual, clear thing. Now the question is: Is the dampening down of telomerase that you see by these other conditions that are not just frank genetic disease sufficient to damp down the stem cells? Its hard to get stem cells from healthy people. We dont have that access. But it is true in the mouse system and in the genetic system of disease. So there is a reason to think that it could be true. Now how much that contributes and how much other things contribute, we dont know. We have got a window on the immune system, which is the blood cells. But it is not telling you about everything else either.

But is the depletion of the telomerase activity secular across all cells of the immune system or is it specific only to stem cells?

We have started looking at that now. We really subdivide the white blood cells into the six classes that we know have measurable and quantifiable very low but measurable amounts of telomerase. Those are the ones we can get. Wed love to look at stem cells, but we cant. So we look at products of them and the different lineages of the leukocyte class of things. We know that each group of cells has characteristic distribution of telomere lengths; we are finding that they have characteristic telomerase [too], very low but its still characteristic. We are just learning our way into the system right now. This is all new. No one thought to do that. We did this because of these associations with disease risk. So now we are going back and trying to get our minds around whats really going on there. First of all, we have to find out if even the quantities are relevant and then doing molecular tests to see how it is being controlled. We will publish this soon, but we havent. Like in [all] science, we are just feeling our way forward all the time.

You have been emphasising that what is observed as telomere shortening and the role of telomerase in human health is an association and not really a causal connection

We know that in rare genetic diseases there is clear causality though these genetic diseases are a more extreme by definition and so they are not in the general population. But when you look at them, they give you an idea of whats happening in a relatively extreme case. When you compare what happens in more common situations with modulated amounts of telomerase action with what happens in extreme cases in genetically engineered mouse models, can you put them all together? We can [then] make a case for causality that when telomerase is down, it does have consequences. But one just wants to be careful not to over-interpret the shades of grey kind of thing. As a scientist, as somebody who is looking at it all, I always want to be careful because biology has a way of turning around and surprising you. So the weight of evidence is pretty much like this: lower [levels of] telomerase could indeed plausibly contribute to things like diseases of aging.