Elizabeth Blackburn (UCSF) Part 2: Telomeres and Telomerase in Human Stem Cells and in Cancer

Welcome to the second part of this three-part lecture series that I’ll be giving on telomeres and telomerase. In part two, I’m going to discuss telomeres and telomerase in human cells, and particularly I’m going to emphasize the setting of cancer cells. Now you may recall from the first lecture that the function of telomerase is to maintain the telomeres and prevent them from shortening as cells divide, because telomere shortening would otherwise occur in the absence of telomerase, to compensate for the shortening processes.

And so, maintaining telomeres allows the cells to keep on dividing. In human cells, we can also see though that telomerase itself is protecting telomeres, and so I want to show you one kind of experiment for that. And the conclusion is going to be that it’s not just the bulk telomere length that matters, but the presence of telomerase can determine whether a telomere is seen by the cell as sufficiently long or not.

The experiment I’ll show you was done in cultured human cells. These were not cancer cells, and these were cells that have normally extremely, extremely low levels of telomerase, effectively, for our purposes, essentially no measurable telomerase activity, and certainly their telomeres are not maintained. Now I’ve shown you in very simple diagrammatic form human telomerase, so this would be the template sequence of the human telomerase RNA, and this is the overhanging G-rich strand. It’s actually usually longer than this, but I’ve just shown it as a simple diagrammatic form here. And this of course is the duplex telomeric DNA that will be consisting of hundreds or thousands of telomeric repeats, as you go in toward the chromosome interior. And just as I showed you for Tetrahymena telomerase, the template region is copied, and so the DNA… for example, a DNA with three Gs, two Ts, and an A, would sit down here on the template, and then nucleotides would added, extending along the template, thereby lengthening the DNA in this reverse transcriptase reaction that telomerase carries out.

So what was done was to compare cells in which telomerase was being expressed or not being expressed, and look at the growth of the cells and the telomere length. Now the protein TERT is the core protein that has this enzymatic activity: the reverse transcriptase activity, and probably other activities, as I will allude to. And so, in these experiments, a particular mutation was made on the TERT protein, it happened to be a very small change of a few amino acids added to the very C-terminus, and what this does is it doesn’t affect the enzymatic activity, but it does affect the ability of this enzyme to elongate telomeres in cells, and it’s called a hypomorph because that refers to the fact that it has an insufficient function, but it showed something useful. Now here’s the experiment, and I’m going to walk you through this rather complex-looking slide.

First of all, I want to show you a growth curve of human fibroblasts in culture. What normally happens is, if we look at the… this is cumulative dilutions, which is just an operational term telling you how many cell divisions are going on, and this is the number of days. So if you culture human fibroblasts, normally what you find is that the cells will continue to multiply for a while, and then they’ll cease multiplying any further, so the curve flattens out, so you see they’ve undergone something like 50 or so divisions. And if you put in a control vector, which would be important, you get the same curve. But if you put in the test vector, which actually is expressing this form of the human telomerase core protein TERT, because the fibroblast cells naturally have enough telomerase RNA and the other components of telomerase, all they’re missing is the TERT, you just have to add this in, and now you can restore telomerase activity.

But what’s very interesting is the telomeres and the cell growth. What you can see is, first of all, the cell growth has been greatly extended. We’ve now made these cells very much elongated in their lifespan compared with the controls or the parental cells. Now let’s look at the telomeres. If we look at the telomeres in these cells, the controls, we find that they’re gradually becoming somewhat shorter, and at this point here, they’ve pretty much ceased to divide, so we’re out here, they pretty much cease dividing. So the telomeres on average are about this long, and the cells have picked up the signal, they’ve said the telomere length is shorter here than here, and they’ve picked up the signal, and they’ve said we’re not going to divide any further.

Now we’ve added the hTERT. Now actually what happened was that, I told you the cells continue to grow a long, long time. The telomeres shorten, and then they steady out at some much shorter length, so through here and through here, they’re actually growing quite well, but they’re going with much shorter telomeres. So in other words, these cells can keep dividing for a long time with very short telomeres, and the difference was they had telomerase being expressed. So we used a trick just to separate out the telomere lengthening property of telomerase from its ability to stabilize telomeres, and we saw that in fact if you have this telomerase present throughout, even though the telomeres maintain short, they are perfectly stable, and the cells can keep dividing. So this is the second piece of evidence, I showed you the first piece of evidence for you in yeast systems in the first part of this lecture series, and now this is the second piece of evidence that telomerase is having a protective function, in this case, it’s in human cells, and it’s stabilizing telomeres that otherwise would’ve been too short in its absence.

This is not unique to human cells, it’s been seen in yeast systems as well experimentally. So I just talked to you about normal cells, and I told you that, in those human fibroblasts grown in culture, there’s very little telomerase. So, now let’s talk about where do we normally find telomerase in human cells? Well, if you look in cells, you find that it actually is on at times when the cells are greatly proliferating during fetal development, and it does remain active in certain proliferative cells.

It’s also found active in stem cells, in cells that are activated to proliferate, such as lymphocytes proliferating under the response to, for example, a pathogen. One finds stem cells, for example, in hair follicles; those have telomerase. So one does find telomerase in cells that are stem cells and various sorts of cells that are induced to proliferate. And in fact in most other cells, one can find telomerase, but it’s in very low levels. So initially people thought there was no telomerase in normal epithelial cells or fibroblasts or endothelial cells, but closer scrutiny showed that in fact there were real, definitely low and very downregulated, but real levels of telomerase.

And in the third part of this three-lecture series, I will talk more about the telomerase in the normal cells of people and tell you about some in vivo studies that have been done. Now, cancer cells. Cancer cells are infamous for their ability to keep on multiplying. Now, they can do this for a variety of different reasons. They lose their system of checks and balances that prevent them from overmultiplying, and that’s because of a lot of genetic and epigenetic changes that have taken place in their progression to become a malignant cancer cell. Now, they are immortal cells, and indeed, as you might expect, telomerase is on in these cells, and in fact it’s very high in the vast majority of human cancers, particularly as they’ve got to the invasive stages. And that makes a lot of sense based on what I just told you, because if the cells are to keep on multiplying, then they have to keep on replenishing their telomeres. Now I’ll you some more recent results in the last few years, which also suggest telomerase may be doing other things in human cancer cells. It is certainly maintaining the telomeres, and that is important, but there may be other functions as well.

So I’ll tell you some newer findings about that. So, let’s just recapitulate what I said about where we find telomerase in humans. So it keeps telomeres elongating and replenished, and therefore cells can keep dividing in stem cells and, of course, I didn’t mention germ cells. Now of course we wouldn’t be here if we didn’t have maintenance of telomeres from generation to generation. Telomerase indeed is active in germ cells, as well as stem cells of various kinds. As I said, it’s detectable in many normal cell types, and highly active in the great majority of human cancers. And by the way, in some of those human cancers in which you don’t find the high telomerase, one actually often finds this ALT mechanism, but it’s only a particular subset of cancers in which one finds ALT being a prominent means of maintaining telomeres. The great majority of the common human tumors have highly active telomerase. So, telomerase is highly active in human tumors. So one could imagine inhibiting telomerase, and this might be a good target for trying to inhibit the growth of cancer cells, if you could inhibit telomerase in cancer cells.

And so in investigating this, some interesting things emerged. So let’s just think about what is expected to happen if you don’t have telomerase in the cancer cells. Well, as you know, cells multiply, and their telomeres will progressively become shorter and shorter if there isn’t telomerase to counteract that shortening, and so eventually when the telomeres get too short, the cells would eventually cease to divide, and the cells respond to those short telomeres by either what’s call the “senescence response,” in which the cells simply won’t replicate their DNA anymore, or a cell death response that can include an apoptotic response, which involves an active cell suicide program that can be induced by dysfunctional telomeres. Whether it’s senescence or cell death does depend upon the cell type. So, the simple prediction, diagrammatically, would be, if you didn’t have telomerase, the cells now would have shorter and shorter telomeres, and after some number of divisions, eventually there would be cell death. Without telomerase, it’s been observed experimentally, typically human cells lose their telomeric DNA at this kind of rate. I’ve put 150-200 base pairs per cell division; that’s seen in a number of cultured cells and also in some cancer cells in which telomerase has been inhibited.

Now, I told you that human telomeres are typically made of hundreds, even thousands, of copies of telomeric repeats, they’re thousands of base pairs long, so at this rate, it’s going to take quite a lot of cell divisions before the telomere gets short enough that the cells will have this response. So that in fact is the prediction, and if you inhibit the telomerase enzyme using, for example, a small molecule inhibitor that inhibits the catalytic function of telomerase that prevents it from carrying out that DNA polymerase reaction by the reverse transcriptase mechanism, if you inhibit telomerase in such a way, indeed this is the observation.

There’s a gradual shortening of telomeres, and eventually the cells cease to divide. So this is seen in cancer cells in culture treated with such an inhibitor. Now I’ve put a noted point up here: but you’re still keeping the telomerase ribonucleoprotein (RNP) level high. You’re not depleting the cells of the enzyme, you are simply rendering that enzyme inactive. And I make that distinction because of the next results I want to tell you about. So, what was observed was quite surprising.

If one depleted telomerase, and the particular way to knock the telomerase RNA down, then one found there was a very rapid effect on human cancer cells. One didn’t see the long delay ensuing before the effect was observed. So, now I’ll tell you about those experiments. So, reminding you again, here’s human telomerase, it’s copying its RNA template, and of course the enzyme has the TERT protein and the telomerase RNA, and it’s the RNA that is going to be depleted in these experiments. How’s the RNA depleted? A now commonly used technique, which is called a knockdown using RNA interference. Now the particular technique was to express from a lentiviral vector, which you can introduce efficiently into cells, a particular sequence which forms a double- stranded RNA, and I’ve just shown the corresponding DNA sequence here, and that double-stranded RNA can interact with a cellular RNA that matches its sequence, and eventually cause the breakdown of that RNA through a complicated process known as RNA interference.

And so, siRNA refers to “short interfering RNA,” because such a short interfering RNA, which involves introducing two strands of RNA complementary to the target RNA, that is what causes the breakdown to occur. Now when we did this experiment, so here’s a control where we’re looking at a reference amount, here’s the telomerase RNA, one found that in fact the method of introducing such a short interfering RNA by this kind of construct here (the details don’t matter), one could in fact quite efficiently knock down the telomerase RNA in human cancer cells grown in culture, for example, in these breast cancer cells grown in culture. And so one could knock it down almost down to about 10-12% of the original level that one sees in the controls. So what happens then? Now, if what I had told you was the case, that we knock down the telomerase, then we would expect to have to wait for a long time for the telomeres to get short enough, if that were the only thing going on, then we would have to wait before we saw any effect.

But right away, there was an effect on the growth of the cells. Now just to put this into perspective here, I’ll just walk you through a couple of graphs. Here’s the controls of two kinds, and this is the number of cells here. And so what you can see is that the cell number goes up and up and up, as you might expect. The cells are dividing once every day or two, and so you can see, after about four or five days after the introduction of the construct that knocked the telomerase RNA level down, that the cells that received this are quite quickly growing more slowly than the controls. So this must be occurring within a couple of cell divisions. And here are some more controls in which, here’s the empty vector, here is the short interfering RNA targeting telomerase RNA, and here’s a control version of that, in which it now no longer can target the telomerase RNA, but everything else is the same, and as you can see it behaves like the controls.

And so a great many control experiments were done to show that this was a specific effect specific to knocking down the telomerase RNA. This was done on bulk, unselected cell populations. The cells were a melanoma cell line that normally has very long telomeres. The bulk, unselected populations is significant because what it meant was that one could put the siRNA, the agent that knocks the telomerase RNA down, into the cells at day zero, and without even any selection, one could get something like 80-90% of the cells that received this construct, and in fact the ones that did grow out were that low percentage that didn’t receive the construct.

So in fact the effect is even stronger than what these curves would indicate, because these ones that are growing out are largely the ones that just didn’t receive the construct. Now, one of the things that is very important in human cells to respond to DNA damage such as certain kinds of telomeric DNA damage is the gene p53. Interestingly, these effects did not require p53. Here is a pair of cell lines that are otherwise isogenic. This is a colon cancer cell line called HCT116, in these cells the p53 is wild-type, here’s the response to knocking down telomerase RNA. In this otherwise isogenic cell line lacking p53 but otherwise the same, this response is quantitatively the same. So this is a different response from a classic DNA damage response, and in fact we did not see DNA damage response genes being induced here. We looked at the telomeres by some molecular probing mechanisms that allow one to see if the telomeres are uncapped, and in fact they were not uncapped either.

So we see, when we knock telomerase RNA down, we rapidly see an inhibition of cancer cell growth, these are cells that normally have very high telomerase. p53 is not required for this, suggesting it’s not a classic DNA damage response. And the telomeres, as far as we can see, are not uncapped, and there’s not DNA damage response. Indeed, the telomeres hadn’t had time to shorten perceptively at all during this short timeframe. So the rapid knockdown doesn’t uncap the telomeres. That’s not the cause of the growth inhibition of these human cancer cells. But interesting things happen very quickly to these cells when you knock down telomerase RNA. I’m going to show you this one experiment in which, in this experiment, melanoma cells were grown in culture, and the RNA level was knocked down by a completely different mechanism, it’s called a “ribozyme,” and the purpose of it is to cleave the telomerase RNA, causing it to break down, and so it has exactly the same effect that I showed you for the RNA interference: you knock down the telomerase RNA level.

A very interesting thing was seen. These are cells that are growing in culture, and these are three flasks of cells that either are control cells that just got the empty vector. The cells are all growing on the bottom of the flask, you can’t see it. We’re just looking at the medium, the broth, the liquid medium in which the cells are growing, and it’s a nice, pinkish color here in the controls. But here, in two separate versions of the cell lines that received the construct that knocked the telomerase RNA levels down, you can see that the medium has turned a dark color.

These were melanoma cells, so it was of course very easy to wonder if indeed these cells were now producing the pigment melanin in high amounts in this and this but not in the control cells. And indeed, analyses showed that that was exactly what was happening. And in fact, a lot of interesting things happened. These cells became more like their normal counterpart cells. It was as though they were more differentiated; they looked more dendritic in form, as though they had become less cancerous.

And indeed their gene expression profiles had changed. So, knocking down telomerase RNA also made these cells less invasive. The more differentiated the cancers are, very typically, the less invasive they are, and in fact, in preclinical mouse model systems used, in fact metastasis was knocked down. And that was seen whether one knocked telomerase RNA down with the ribozyme, or with the RNA interference mechanism, but in melanoma models for cancer, using the experimental mouse system in the laboratory, it was found that the metastasis is decreased when the telomerase RNA is depleted.

So knocking telomerase RNA levels down changed the nature of the cells, and it changed the nature of the cells well before the telomeres became uncapped. So, as I said, when you have plenty of telomerase, you expect cells to be able to multiple indefinitely, and you don’t expect any effects to occur, of removing telomerase, until a long time has elapsed. So, what we see by this abrupt knockdown of telomerase level, by knocking down the telomerase RNA, is we see rapid inhibition of cell growth, p53 was not required, there was no DNA damage response or telomere uncapping, and in fact metastasis is reduced. Metastasis is reduced, what is going on? I briefly mentioned, we looked at the gene expression profiles in these cells, and we found that in fact that rapid knockdown of telomerase RNA and that rapid slowing of the cell growth is accompanied by and presumably caused by cell cycle and tumor progression genes being downregulated. Glucose metabolism is downregulated; cancer cells typically have high rates of glucose metabolism. That becomes downregulated in cells that have less telomerase RNA. And the cells appear more differentiated, making one wonder if there’s a cell differentiation program that’s induced in these cells.

Unexpected changes, many open questions remain, but these kinds of experiments have led to these observations here that have two kinds of implications: One is the scientific implication that telomerase has other functions that are not solely mediated through its adding telomeric DNA to the ends of the chromosomes, and the other implication is that this makes telomerase an interesting target for potential anticancer therapies. And the take-home message that I think is most impressive and unexpected was, there seems to be good reason to think that high telomerase levels are promoting an undifferentiated, “stem cell-like” phenotype, a very unexpected observation. And in fact, now in other systems, I won’t have time in this lecture to go through it, but there’s now evidence that this is really the case, in even noncancerous cells in certain model organisms where this has been studied..

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