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Telomeres and Telomerase

Part 1: The Roles of Telomeres and Telomerase

00:00:03.20 Hello, my name's Elizabeth Blackburn. I'm in the
00:00:06.25 Department of Biochemistry and Biophysics at the
00:00:09.19 University of California, San Francisco. And in this set of
00:00:13.19 lectures, I'm going to talk about telomeres and
00:00:16.08 telomerase. And I'll get to their implications for human
00:00:20.19 health and disease. The first part of this series of three
00:00:26.07 lectures is going to introduce you to the roles of telomeres
00:00:30.18 and telomerase. So, let's begin by focusing in on what
00:00:36.27 goes on at the very heart of a cell. Now if you looked at a
00:00:42.10 cell which is just about to divide, you looked into the
00:00:46.06 microscope and you stained the chromosomes, this is
00:00:48.14 what you would see. Those blue, double sausage-like
00:00:51.25 objects that you can see all over this microscope slide are
00:00:57.17 the human chromosomes, and the DNA has just
00:00:59.24 duplicated, which is why they look double. Now if you
00:01:03.11 look closely, you can see red spots, two red spots at the
00:01:06.15 ends of every double chromosome pair. And these red
00:01:11.15 spots are a molecular probe that's lighting up the telomeric
00:01:15.05 DNA that's found in common at all of the chromosome
00:01:18.25 ends. And I'm going to tell you a lot about that telomeric
00:01:23.04 DNA. So why are telomeres important? Their role is to cap
00:01:30.14 off the ends of chromosomes. So that's a simple concept,
00:01:35.26 but we can dive down into it further and think a little bit
00:01:38.17 more about what that actually means. So when we think
00:01:42.07 of the end of the DNA, there can be two kinds: There
00:01:45.09 can be the natural end of the DNA, such as the ones we
00:01:48.07 see here, the ends of the chromosomal DNA; and there
00:01:52.12 can also be DNA breaks. Now, the job of a cell is to seal
00:02:01.05 up any DNA breaks that happen by accident. And so a
00:02:05.03 major part of this capping function, which is one of the
00:02:09.04 aspects of telomere functions, is to prevent the telomeres
00:02:13.23 from undergoing those very DNA transactions of very
00:02:17.11 DNA reactions that are undergone by a broken DNA end.
00:02:24.00 So if you have a break in the DNA, it can be sutured
00:02:27.14 together by, for example, recombination or just simply the
00:02:31.21 two broken ends can be ligated right back together by
00:02:34.25 end-to-end fusions. Also, such DNA breaks are subject to
00:02:40.15 degradation. Now the telomere protects, it caps, the end
00:02:45.05 of the chromosome and protects against all of these kinds
00:02:48.13 of things that would normally happen to a broken DNA
00:02:52.12 end. How does it do that? The DNA sequences that you
00:02:58.08 find at the ends of chromosomes, repeated over and
00:03:01.15 over, are fairly similar in nature to each other. They're
00:03:06.11 relatively simple sequences, and they're too simple to
00:03:09.24 code for any proteins. They're not genes in the sense of
00:03:15.09 coding for any proteins or RNAs. In humans for example,
00:03:21.20 this repeated sequence is found up to a few thousand
00:03:24.14 times, tandemly repeated over and over at the ends of the
00:03:29.08 chromosomes. Another feature of the chromosomal DNAs
00:03:34.06 is that, of course, unlike most of the DNA of a
00:03:38.00 chromosome, which is duplex DNA, double-stranded
00:03:40.11 DNA, the very end is single-stranded. And in fact the
00:03:44.26 DNA strand is oriented 5' to 3', going toward the end of
00:03:50.03 the chromosome. And that turns out to be important. So,
00:03:58.12 we have now the telomere structure to begin with: We
00:04:01.27 have again, if we blow up the end of a chromosome, we
00:04:05.23 have these highly repeated sequences made up of a G-
00:04:10.12 rich sequence that's the repeat unit repeated over and
00:04:13.13 over again. As I said, it doesn't encode any protein
00:04:17.13 sequences, but each of these repeated sequences is like
00:04:23.05 a little attractive magnet for specific proteins that bind
00:04:27.25 sequence specifically to the telomeric DNA. They bind to
00:04:31.22 the telomeric repeats for the double-stranded portion, and
00:04:35.27 some of them bind the single-stranded portion, and that's
00:04:39.06 actually the G-rich strand that's forming the overhanging
00:04:45.11 end here. These together make some form of higher order
00:04:53.02 architecture we don't understand. We understand a lot
00:04:56.13 about the protein-DNA interactions, some of the molecular
00:04:59.11 details of that. Some of the details of the protein-protein
00:05:02.19 interactions in this complex, but we don't really
00:05:05.07 understand the higher order structure, so that's still a
00:05:07.22 challenge in the field. So I've just shown you a functional
00:05:14.04 telomere, but if telomeres cease to function... and we use
00:05:18.10 the term "telomere dysfunction" to just describe that
00:05:21.21 general state of the telomere that is not carrying out those
00:05:25.04 capping functions and other functions that I will get to...
00:05:30.07 there are a couple of different ways this can happen. The
00:05:33.09 first is if the tract of telomeric repeat is simply too short,
00:05:38.27 there's just not enough of the length of the repeats to form
00:05:44.27 a nice, long array that can form this higher order structure
00:05:48.27 that's necessary. This kind of dysfunction caused by the
00:05:54.27 shortening of the telomere, that can happen naturally, and
00:05:59.28 it does, and we'll get back to that in a moment, because
00:06:02.24 this is going to be an important part of these lectures, and
00:06:05.28 in fact it'll be really the focus of the third lecture in this
00:06:10.00 series. The other way that telomeres can become
00:06:15.07 dysfunctional: If for one reason or another, experimentally
00:06:19.12 induced, most commonly, one of these proteins cannot
00:06:23.27 bind correctly to the telomeric DNA; if its binding is
00:06:27.19 disrupted through some molecular intervention or other...
00:06:32.22 in both cases, cells sense and respond to this state of
00:06:40.03 telomere dysfunction. Now indeed, cells have a lot of very
00:06:46.22 strict regulatory reactions to the lack of proper telomeric
00:06:53.03 DNA. And the consequences for the cell is that, usually
00:06:59.06 this state of the cell's telomere dysfunction, through one
00:07:05.02 reason or another, will mean that the cell will cease to
00:07:10.20 divide. So this limits cell renewal capability if this happens
00:07:15.16 to one or more of its telomeres in the cell. If by chance the
00:07:20.20 cell does continue to multiply, now those telomeres
00:07:26.21 become subject to the very kinds of fusions (the DNA-
00:07:31.28 joining events that I told you telomeres shouldn't allow to
00:07:35.11 happen), and that can lead to genomic instability,
00:07:39.21 because the end-to-end joining of telomeres to
00:07:42.17 themselves (other telomeres, that is) or to broken DNAs,
00:07:47.00 that can cause the chromosomes, which fuse to each
00:07:52.07 other, to tear themselves apart as the cells divide, leading
00:07:56.09 to genomic instability. So clearly telomere function is very
00:08:02.20 important for cells. And in fact, one of the consequences
00:08:07.10 of genomic instability in human cells is that the cells can
00:08:11.13 become cancerous. I'm just going to show you a picture.
00:08:17.26 If you look under a microscope of some cells in which
00:08:22.20 we've disrupted one of the telomeric proteins, what you
00:08:25.22 can see is... remember I told you the blue, double things
00:08:30.01 are the chromosomal DNAs... and look, here's a
00:08:32.29 chromosome here in which there's been a telomere
00:08:35.19 fusion. So here's the two telomeres at the end, here's the
00:08:38.25 other two all the way here, but there are fused telomeres
00:08:43.27 here. So this is now a chromosome that has two
00:08:47.15 centromeres, it's got a centromere here, it's a got a
00:08:50.02 centromere here, and if those two centromeres try to pull
00:08:54.05 apart in a cell that is dividing, the chromosome will get
00:08:59.15 ripped apart. Here's another example of such an end-to-
00:09:04.22 end fused chromosome. This kind of change can happen
00:09:09.05 if you disrupt the telomeric integrity by, for example,
00:09:13.05 disrupting the binding of proteins. The other kind of
00:09:18.23 function, it's related, but we can distinguish it, the other
00:09:23.02 kind of function of telomeres is that they have to allow for
00:09:27.13 the complete replication of the telomeric DNA. So what's
00:09:33.07 the issue here? Well, the mechanism of DNA replication,
00:09:40.12 the machinery of DNA replication, has a particular quirk to
00:09:44.23 it. It's very good at faithfully copying almost all the way
00:09:49.19 along the length of the chromosomal DNA (or any linear
00:09:53.17 DNA), but the make-up of the DNA replication machinery
00:09:59.00 is such that is cannot copy the very, very end of the linear
00:10:04.28 DNA, such as a eukaryotic chromosomal DNA. Now the
00:10:10.11 predicted and observed consequence of that inability is
00:10:14.22 that, each time the DNA replicates, which is has to do as
00:10:20.01 the cell divides, and then the cell divides, the daughter
00:10:24.00 DNAs are predicted to become shorter and shorter and
00:10:27.13 shorter. And this is just a simple consequence of the
00:10:32.15 nature of the DNA replication machinery that is otherwise
00:10:37.00 so good at replicating all the way along all the length of
00:10:41.07 the chromosomal DNA. But the very ends cannot be
00:10:45.09 completely replicated without some form of compensatory
00:10:49.20 mechanism. So what's the consequence of this loss?
00:10:53.20 Well, obviously, something has to compensate in the long
00:10:56.06 run, otherwise, we wouldn't be here. But even on a
00:10:59.29 shorter timeframe, as cells divide and divide, the DNA
00:11:03.08 gets shorter and shorter, one might predict that there
00:11:05.24 would come a point when there wouldn't be enough of
00:11:10.10 something or other at the end that then the chromosomes
00:11:14.19 would no longer be able to support cell division, and the
00:11:18.00 cells might eventually undergo what's called senescence.
00:11:22.00 And indeed James Watson in 1972, just considering the
00:11:26.02 mechanism of DNA replication, proposed this constant
00:11:30.15 shortening problem, and Olovnikov around the same time
00:11:36.20 proposed that in fact perhaps such loss of terminal DNA,
00:11:41.22 without knowing at that stage what the molecular nature
00:11:44.07 of the terminal DNA was, Olovnikov proposed that
00:11:47.12 perhaps such gradual loss could be something that
00:11:50.20 underlies the eventual senescence of cells that is seen
00:11:54.14 sometimes when, for example, human cells are grown in
00:11:57.08 culture. This was a prescient idea because, in fact, this
00:12:02.04 indeed has been found to be one of the causes of why
00:12:07.17 human cells cannot replicate themselves, the cells cannot
00:12:12.18 proliferate, indefinitely in culture. So, shortening of
00:12:19.26 telomeric DNA is something that will be problematic for
00:12:25.12 cells. How is this problem solved? Well, it's solved by an
00:12:33.01 enzyme called telomerase. So I'd like to introduce you to
00:12:37.12 telomerase now, and how it was found. Telomerase was
00:12:46.06 sought because there was a set of accumulating
00:12:49.24 observations on telomeric DNA in cells, the telomeric
00:12:54.24 DNA as it was in cells in vivo, that couldn't be readily
00:12:58.22 explained by what was currently known about DNA
00:13:02.12 replication or DNA recombination or other kinds of DNA
00:13:06.22 reactions at the time, which was the late 1970s, early
00:13:10.21 1980s. And let me give you some examples of such
00:13:15.14 puzzling observations. Well, the first one was that in the
00:13:21.26 ciliated protozoan Tetrahymena, which has a lot of very
00:13:24.26 small mini-chromosomes and is therefore amenable to
00:13:28.02 molecular analyses of its telomeric DNA by direct
00:13:31.09 methods, the telomeric repeat sequences (remember I told
00:13:36.05 you about the repeated sequences repeated over and
00:13:38.16 over at the ends of chromosomes)... the repeat sequence
00:13:41.28 in this organism, G4T2 repeats, were heterogeneous in
00:13:48.05 their number, in different molecules in a population of
00:13:53.01 otherwise homogeneous cells. And heterogeneous in this
00:13:57.20 setting here is meaning, these were different in number,
00:14:04.04 so some copies of the mini-chromosome would have
00:14:07.07 twenty repeats on the end, some would have 50-some,
00:14:09.09 49-some, 82-some, 53... they all had different numbers of
00:14:13.16 repeats in this sort of more-or-less normal distribution. So
00:14:18.18 that was very surprising, because if you look across at the
00:14:21.15 internal region of a DNA, such as a chromosomal DNA, if
00:14:25.20 you look at one cell compared with another in a
00:14:29.05 population of cells from, say, one organism, it should
00:14:32.08 always be an identical sequence. So here were different
00:14:35.21 numbers of repeats at the ends of different molecules in a
00:14:40.15 population of cells that should have otherwise been
00:14:43.07 homogeneous, and were homogeneous in their internal
00:14:46.09 regions of the chromosome. Now, a second kind of
00:14:50.17 observation was a somewhat more complicated one, and
00:14:55.14 it means that I have to just take a moment to tell you
00:14:59.12 about the lifecycle of a particular group of ciliated
00:15:02.23 protozoans, which the species Tetrahymena in which
00:15:07.08 these G4T2 repeat tracts were found to be the telomeric
00:15:10.26 tracts. The Tetrahymena cells go through a lifecycle
00:15:15.24 stage where they have a somatic nucleus which
00:15:20.09 undergoes developmentally controlled fragmentation. And
00:15:24.16 fascinatingly, telomeric DNA sequences were added
00:15:27.29 directly to those freshly formed DNA ends, making from
00:15:33.27 longer chromosomes a series of shorter mini-chromosomes.
00:15:38.25 How did that telomeric DNA get added to
00:15:41.15 the ends? It wasn't clear. The third observation came
00:15:48.11 from observing the cells of an organism which causes
00:15:52.09 sleeping sickness, a single-celled parasitic organism
00:15:55.24 called a trypanosome. And these were being propagated
00:15:59.07 in the laboratory setting, and what was found was that the
00:16:04.05 telomeric DNA restriction fragments, the end fragments of
00:16:08.05 the chromosomes in these organisms, were gradually
00:16:11.09 getting longer and longer and longer. And this didn't look
00:16:14.10 like, say, recombination, and certainly was not expected
00:16:18.01 for normal, as one thought about it, DNA replication. The
00:16:23.27 fourth observation was again something that came out an
00:16:29.00 experiment that I'll have to explain. Now one could put
00:16:33.07 circular plasmids into yeast cells, and those plasmids are
00:16:37.01 linearized, essentially they're unstable, they get gobbled
00:16:40.16 up or, rarely, will recombine into the chromosomes and
00:16:44.12 thus be preserved. But what was found was that if one
00:16:48.10 simply grafted onto the ends of such a linearized and
00:16:52.24 therefore normally very unstable yeast plasmid, if one
00:16:56.24 grafted onto its ends Tetrahymena telomeric DNA
00:17:01.15 fragments (the telomeres of Tetrahymena mini-
00:17:03.24 chromosomes) and introduced those into cells... so the
00:17:08.01 grafting was done in vitro with enzymes and the purified
00:17:11.09 DNA molecules, and then the resulting hybrid of the yeast
00:17:17.05 linearized plasmid and the telomeres added onto its end,
00:17:21.29 that was introduced into the yeast cells, those were
00:17:25.00 maintained in yeast cells as linear mini-chromosomes, and
00:17:28.12 yeast telomeric DNA repeat were grafted on somehow
00:17:34.11 inside the yeast cells, to the ends of the Tetrahymena
00:17:39.02 telomeric repeats. And that didn't look like a reaction one
00:17:43.11 would expect from standard known models of DNA
00:17:46.26 replication or recombination. All of these suggested that
00:17:52.03 perhaps there was some capability of cells to add
00:17:57.16 telomeres. And that idea, in a conceptual way, was given
00:18:04.22 some force by an observation made by the noted
00:18:09.25 geneticist, Barbara McClintock, who worked with maize
00:18:13.06 (corn), and she noted that a particular maize mutant stock
00:18:17.28 lost the capacity to do something that is normally found in
00:18:23.24 normal, wild-type maize. In such wild-type maize, if a
00:18:28.28 chromosome is broken by, for example, exo-radiation or
00:18:32.15 some mechanical rupture at a particular stage in
00:18:37.07 development, then that broken end can be, as she
00:18:40.18 described it, "healed." It becomes a normal, stable
00:18:43.29 telomere. Nobody knew the molecular basis of that. And
00:18:47.29 she found a mutant that had lost that capacity. And when
00:18:51.04 you see a mutant where something is changed, you have
00:18:53.17 a feeling that that's reflective of a cellular process that
00:18:57.21 can take place, and that it's not just by chance that this
00:19:01.08 healing event was taking place. All of which, then,
00:19:07.23 focused in on the question of: Was there a new enzyme
00:19:10.02 that worked in cells that could extend telomeric DNA? So
00:19:14.17 we sought such an enzyme in the early to mid-1980s. And
00:19:19.24 we used for that purpose the single-celled, ciliated
00:19:24.18 protozoan Tetrahymena thermophila, shown in this
00:19:27.18 scanning electron micrograph here. And you can see the
00:19:31.02 cilia are on the cell surface. This experimental system was
00:19:34.26 chosen because the organism contains large numbers of
00:19:40.00 very short mini-chromosome, therefore, large numbers of
00:19:44.15 telomeres, and therefore, one would reason, perhaps if
00:19:49.03 there were such an enzyme that existed that could add
00:19:53.03 telomeric DNA to the end of chromosomes or to
00:19:56.25 preexisting telomeres, then this would be a potentially
00:20:00.05 good source for it because there's a lot of telomeres in
00:20:03.28 this organism. And indeed that proved to be the case.
00:20:08.18 And Carol Greider, my then-graduate student, joined the
00:20:12.11 lab in 1984, and here's a picture of Carol, freshly from
00:20:16.10 visiting Southern California, and looking at an
00:20:20.29 autoradiogram shown with this x-ray film here in the lab.
00:20:26.17 And we together found this enzyme telomerase. Now let
00:20:32.15 me show you briefly what we did. We took a mimic of that
00:20:37.18 overhanging G-rich strand DNA that's normally found at
00:20:41.11 the ends of chromosomes, in the form of an oligonucleotide,
00:20:45.18 and here it is shown here. And I've just
00:20:47.13 colored the bases different colors so that we can see
00:20:50.22 them easily. And here's its 3' hydroxyl end. We mixed it
00:20:55.28 with an extract of Tetrahymena cells, and this worked
00:20:59.13 particularly well at the stage in development that I
00:21:03.22 mentioned to you earlier... that stage at which telomeric
00:21:07.20 DNA is added to freshly broken ends of chromosomes.
00:21:12.09 We reasoned that that would be a particularly good time
00:21:15.29 to find such an activity, thinking it might be likely to be
00:21:20.16 induced or present in perhaps larger-than-normal
00:21:23.25 amounts, because this would be a time when there'd be a
00:21:27.02 demand for such an enzyme. All of this was hypothetical
00:21:30.00 at the time, but by putting in an appropriate mix of things
00:21:33.17 that are often added to make polymerases happy (such
00:21:39.02 as magnesium ion), and just by using two nucleoside
00:21:43.17 triphosphate precursors, dGTP and TTP, we were able to
00:21:48.21 find that the telomeric DNA indeed was added to the end
00:21:53.02 of such an oligonucleotide. In fact, in the test tube, a
00:21:56.09 large number of repeats could be added. So we get a lot
00:22:00.22 of repeats added, through eventually something limits the
00:22:06.18 addition. So this is what telomerase did. Now how is it
00:22:14.16 doing that? It was adding a given sequence. The first
00:22:18.09 clue came from the observation... and here I'm just
00:22:22.01 showing you a nice picture of the products run on a DNA
00:22:27.10 sequencing gel, just to give you feel for what it looks like.
00:22:30.14 We'll just look at one of these groups, so here are
00:22:33.13 different time points, increasing number of minutes after
00:22:37.09 incubating this input primer with dGTP and TTP. The
00:22:41.06 dGTP was radiolabeled, so we're just looking at an
00:22:43.26 autoradiogram. Every time we see a labeled band, this is
00:22:47.22 a product that's been added, so the input would run here.
00:22:50.20 If you added one, two, three, four nucleotides, you could
00:22:53.11 see you'd get longer and longer bands. And what you can
00:22:56.20 see is that there is kind of a striped pattern. Every six
00:23:01.05 nucleotides, there was a pause in the addition, and so
00:23:04.16 you could see a pattern of six nucleotides being added,
00:23:09.03 and you could see more and more repeats, as shown by
00:23:11.20 the bands getting higher and higher and higher, were
00:23:14.18 being added with time. Now, certain clues came. Not any
00:23:21.27 nucleotide could work. So, two examples of the telomeric
00:23:27.01 DNA, where we've got a permutation ending with four Gs
00:23:30.12 here, or a permutation ending with two Ts here. These
00:23:34.26 were each competent for addition by this activity.
00:23:41.04 However, the complementary strand, and again, I've just
00:23:44.07 colored the bases distinctively and given you two
00:23:47.01 examples... the complementary strand oligonucleotides
00:23:50.25 were not competent for such addition. Another interesting
00:23:59.04 clue came when we looked in more detail at different
00:24:02.16 oligonucleotides that could act as primers. Now, we had
00:24:10.01 found, as I said to you, that when you put Tetrahymena
00:24:13.20 telomeric repeats into yeast cells, live cells, then yeast
00:24:18.12 repeats were added somehow, it was unknown then, to
00:24:22.15 the Tetrahymena telomeres. And the yeast repeats have
00:24:27.06 a different sequence, and I've shown you a little segment
00:24:29.06 of it. It's T, G, sometimes one G, sometimes two,
00:24:34.13 sometimes three Gs. So we wondered if the converse
00:24:38.23 would be the case; if we added a yeast primer to a
00:24:43.14 Tetrahymena extract, now in vitro, would we see
00:24:48.04 Tetrahymena sequences added to the yeast telomeric
00:24:52.19 oligonucleotide. And indeed, we did see such addition.
00:24:58.26 And again, large numbers of repeats could be added to
00:25:02.14 such a primer quite efficiently. Now we also noticed
00:25:08.19 another interesting thing about the reaction. When we
00:25:12.21 had a primer that ended with four Gs, what we found was
00:25:17.15 that first two Ts were added, and then four Gs, two Ts,
00:25:20.15 and so on. When we had a primer that ended with three
00:25:23.06 Gs, first a G was added, and then the two Ts, in other
00:25:27.19 words completing this run of four Gs. And I haven't shown
00:25:30.26 it, but if you had a primer that ended in two Gs, then GG,
00:25:35.07 and then TT. So this suggested that perhaps something
00:25:39.22 was aligning this very first portion, the portion of the
00:25:46.29 reaction that's taking place, adding to the very 3' end of
00:25:50.09 the primer, that perhaps something was aligning this, in
00:25:53.29 the enzyme somehow. Because in other words, if you
00:25:58.10 lined it up like this, then everything was lined up. What
00:26:01.08 could be doing this? So was there something that aligned
00:26:04.28 the product-forming part of this enzyme? Remember this
00:26:10.17 was very mysterious at the time. So in fact we looked and
00:26:15.07 found that, indeed, as I said, you had four Gs, and you
00:26:19.25 added two Ts. If you had three Gs, one G was added.
00:26:23.08 Two Gs, and you'd have two Gs added, and then the two
00:26:25.14 Ts. And if you had one G, then it would be three Gs, and
00:26:29.08 then the two Ts. So in fact, there was something that was
00:26:31.27 aligning how the next repeats were added, dependent on
00:26:36.10 the 3' end of the primer. The result of all of this kind of
00:26:44.02 analysis led us to the idea that there was in fact a
00:26:47.15 template within the telomerase, and to our surprise, this
00:26:52.07 template turned out to be made of RNA, an RNA that is
00:26:56.25 actually built in to the telomerase complex. And this
00:27:01.04 template is a short portion of this RNA, which otherwise
00:27:05.26 has a lot of other structure which is built into the enzyme
00:27:09.20 particle, which contains also a protein, which is called
00:27:15.18 TERT. So what telomerase does is it takes that single-
00:27:20.18 stranded G-rich strand, it aligns the 3' end nucleotides by
00:27:27.07 Watson-Crick base pairing onto the template sequence,
00:27:31.29 so here's the example where we have two Gs at the end
00:27:35.05 of the primer. It aligns it on this part of the sequence, and
00:27:39.20 then it polymerizes, one at a time, each of these
00:27:44.29 nucleotides onto the DNA end, extending the DNA end
00:27:53.02 by copying this template. And so now, you end up with
00:27:58.23 longer DNA. So telomerase is a unique polymerase, it's a
00:28:06.22 reverse transcriptase by the definition, that is, copies RNA
00:28:12.18 into DNA. It's unique because the RNA component is
00:28:20.07 actually intrinsically built into the telomerase ribonucleoprotein
00:28:24.13 particle. It has to be built in for that
00:28:29.13 templating to take place. And indeed the enzyme is truly a
00:28:34.07 ribonucleoprotein enzyme, we believe. And unlike, for
00:28:39.26 example, the reverse transcriptases that copy, say, the
00:28:43.03 HIV viral genome, which is a genome thousands of
00:28:47.27 nucleotides long, a very complicated sequence which
00:28:51.14 encodes proteins, this enzyme copies very short
00:28:56.25 sequences over and over again. And it does it by this
00:29:00.26 process of aligning the DNA end on the template, and so
00:29:08.19 just to complete the thought here, here's the template. If
00:29:11.28 we polymerize all these together onto the DNA end, now
00:29:15.14 you'll have a new DNA end that ends with TTG, and that
00:29:19.26 will realign in the next round back in this AAC, for another
00:29:24.12 round of synthesis, and this can go on in the test tube for
00:29:28.07 many repeats. I've told you about some of the enzymatic
00:29:32.13 properties of telomerase, but what good is telomerase for
00:29:37.03 cells? Well, the answer came by manipulating telomerase
00:29:42.25 in Tetrahymena, the organism in which it was first
00:29:46.14 discovered. So if you remember, telomerase is adding
00:29:52.25 telomeric DNA to the ends of chromosomes, and so the
00:29:59.19 question was: What happened if it couldn't do that? So
00:30:03.17 we looked in Tetrahymena, which, as I said, was a good
00:30:07.03 source of telomerase, and another feature of these
00:30:10.23 organisms that I didn't tell you was that, if you grow them
00:30:14.00 in culture in the laboratory, they're effectively immortal, so
00:30:17.15 long as you keep them fed and under good conditions,
00:30:20.12 they can just propagate and propagate and propagate,
00:30:22.27 seemingly forever. And they have plenty of telomerase.
00:30:29.19 We manipulated the telomerase RNA. Now, we made
00:30:33.14 some changes in the RNA, and I won't go into the details
00:30:36.20 of it, but the effect of such small changes in the RNA I've
00:30:42.17 shown diagrammatically here. The telomeric DNA repeat
00:30:46.25 sequences, which as I said consist of multiple repeats at
00:30:51.17 the ends of chromosomes normally, as the cells went
00:30:56.17 through cell divisions, the telomeres progressively got
00:30:59.11 shorter and shorter, and after about 20 or 25 cell
00:31:02.20 divisions, the cells ceased to divide. So in other words,
00:31:07.07 when we inactivated telomerase, over the succeeding
00:31:11.16 cell divisions the telomeres progressively shortened, so in
00:31:15.18 effect when the cells ceased dividing, they'd become
00:31:19.07 mortal. From being immortal, they'd become mortal. And all
00:31:23.22 we had done was to inactivate telomerase by this small
00:31:28.23 change, so like a little stiletto stuck at the heart of
00:31:31.25 telomerase, we killed the enzyme very surgically, and we
00:31:35.08 were able to make immortal cells become mortal. So, the
00:31:40.17 conclusion is that the telomerase maintains the ends of
00:31:43.24 chromosomes. Telomeres are replenished by telomerase
00:31:47.07 as they keep dividing. And in fact, that continuing
00:31:51.22 replenishment, even in the face of the continuing
00:31:55.01 shortening processes that take place, can compensate
00:31:58.20 for those shortening processes and allow the cells to
00:32:01.15 keep on dividing. So that's what telomerase does for
00:32:05.28 cells. And that's the important message: Telomeres are
00:32:10.18 replenished by telomerase. Now, let's go into a more
00:32:16.03 detailed experiment in yeast cells. If we look at a culture
00:32:20.07 of yeast cells and we plate them out on a plate in a
00:32:24.03 laboratory... all those little white spots are cells that have
00:32:28.04 grown up from individual cells distributed on the plate,
00:32:30.24 they've been distributed on kind of a V-shape pattern
00:32:32.26 here, a triangular pattern. So, the cells are growing well
00:32:37.11 normally. Now if we delete one of the genes for
00:32:40.16 telomerase, either the RNA structural gene or the protein
00:32:43.26 structural gene for the core of the enzyme, now just as I
00:32:48.20 described for you in Tetrahymena, the cells divide and
00:32:54.16 divide, and the telomeres progressively get shorter and
00:32:57.03 shorter, until they get to a point where most of the cells
00:33:00.03 completely cease to divide. And so now they're no longer
00:33:04.27 capable of producing a rich growth of colonies on such a
00:33:09.29 plate. See, there's very little growth here. And we call that
00:33:13.27 senescence. So taking away telomerase is a bit of a
00:33:17.23 delay as the cells go through about 50 or so cell
00:33:20.06 generations, 50 to 80, and then the cells' telomeres get
00:33:24.05 too short, and they undergo senescence. We've
00:33:27.12 scrutinized more closely what happens to cells at this
00:33:31.09 point. Interestingly, a few cells do survive, and in fact,
00:33:42.09 those cells eventually become quite capable of producing
00:33:46.26 good growth on a plate, but they're different. These cells
00:33:51.05 now have very heterogeneous and long telomeres. I'll
00:33:55.01 show you these in a more graphical form in a moment. A
00:34:01.17 gene required for this to happen, for such "survivors," as
00:34:06.03 we call them, to appear, is the gene RAD52. For at least
00:34:13.05 one of these pathways, the RAD50 gene is also required.
00:34:17.11 What are RAD52 and RAD50? These are in the
00:34:22.07 homologous recombination pathway. They're needed for
00:34:27.02 recombination and therefore, to get survivors required
00:34:30.28 recombination. Similar phenomena have been seen in
00:34:35.16 mammalian cells. They've been less well characterized
00:34:38.16 genetically, but similar survivor cells have been seen, and
00:34:42.23 they're called ALT cells in mammals, for "alternative
00:34:47.18 lengthening of telomeres"... ALT cells. So, without
00:34:54.10 telomerase, most cells go through senescence, but rare
00:34:57.28 cells can survive. So, that is another way that
00:35:04.21 chromosomes can survive, but interestingly enough, ALT
00:35:10.04 is not normally seen in most normal cells. Now we
00:35:16.17 wondered why, because on the face of it, it looks as if
00:35:20.05 these cells are growing perfectly well. Although if you look
00:35:24.06 more closely, you will see that some of the cells are not
00:35:27.04 living well, but the great majority can survive with these
00:35:30.14 very long telomeres, that they can keep recombining, and
00:35:34.03 keep the population up that way. We asked what's
00:35:38.01 different. So first of all, we quantified very carefully the cell
00:35:43.19 growth, so this is just one way of showing it. The colony-
00:35:46.26 forming units, which is a measure of viability, as the cells
00:35:51.21 went through the progression of shortening telomeres,
00:35:54.17 getting to this point when they get to senescence, and
00:35:56.14 then those rare survivors now overgrew, and now you
00:36:00.29 can see them taking over and becoming the population.
00:36:03.23 So they're growing again quite well. And we looked at the
00:36:07.23 gene expression profile in such cells. First of all we looked
00:36:11.19 at the telomeres, just to make sure that the telomeres
00:36:14.20 were doing what I showed you diagrammatically before,
00:36:17.28 yes they are, so here's the telomeres. And this is what's
00:36:20.20 called a Southern blot, where we're probing with a
00:36:23.00 telomeric DNA sequence. And the most important are
00:36:26.13 these bands down here, that's the easiest to look at
00:36:28.21 initially, because this is a lot of the telomeres. You can
00:36:31.19 see they're of a certain length, and as they get shorter
00:36:34.11 they run faster and faster in the gel, so you can see
00:36:37.07 they're getting shorter and shorter with these successive
00:36:39.21 days of passaging of cell divisions without any telomerase
00:36:44.13 present. Now, there are very few cells here actually, but
00:36:49.09 we loaded enough DNA so you can see what little
00:36:52.11 telomeric DNA there is. And then the cells start growing
00:36:55.00 well again, and what you see is now the telomeres, the
00:36:57.29 pattern has changed, they're very heterogeneous, and
00:37:00.19 there's much longer telomeres, you can see they're longer
00:37:03.18 than these ones here. So that's the telomeric profile. Now,
00:37:13.20 let's look at the gene expression profile. This is what's
00:37:18.01 called a microarray experiment, and in this experiment,
00:37:22.27 one compares the pattern of gene expression by looking
00:37:26.07 at the messenger RNA levels for all of the genes in the
00:37:30.05 genome. And one asks, does a particular gene,
00:37:34.11 compared with a reference, which is right at the
00:37:36.17 beginning, when everything is growing well, does a
00:37:38.26 particular gene's level of expression become higher or
00:37:42.09 lower. If it becomes lower, it gets greener and greener.
00:37:46.12 And so each of these going across is a time point, each
00:37:57.23 line in the column represent a single gene, and each of
00:38:01.05 these horizontal areas represents the day at which the
00:38:08.13 RNA was taken out of the cells and analyzed in this way.
00:38:11.26 And so what we find, and it's all compressed and you just
00:38:14.23 need to look at the pattern, was that about 650 genes
00:38:19.07 changed their expression, and they got either lower levels
00:38:23.22 of expression (and we'll talk about this in more detail, and
00:38:27.20 that's shown in the green) or higher levels of expression
00:38:31.09 (and that shows up as red). Now right away what you can
00:38:35.15 see is that the maximum changes occurred right when the
00:38:38.12 cells were approaching and into senescence. Six-
00:38:43.23 hundred and fifty genes is something like 10% of the
00:38:48.09 genes in the genome, which is about 6000 to 7000
00:38:52.22 genes. So about 10% of the genome shows a change in
00:38:58.03 expression, particularly at senescence, but also, as I'll
00:39:03.05 show you, some genes remain changed even when the
00:39:08.03 cells are growing well, maintaining their telomeres through
00:39:11.26 this recombination mechanism. So one can analyze all the
00:39:17.02 patterns of genes whose expression has changed and
00:39:21.00 compare them with known patterns of gene expression
00:39:24.20 changes that have been looked at in a variety of
00:39:28.00 experimental settings by the many people in the yeast
00:39:33.05 genomics and gene expression community, who've
00:39:36.17 looked at gene expression changes. So one can
00:39:38.28 compare what we see here with what's been seen in
00:39:42.04 other experiments done by many other groups looking at
00:39:46.00 many other conditions of changes to the cells in yeast. So
00:39:53.19 what we found was that in fact, this pattern we observed
00:39:57.04 was unique, and we gave it a name: the telomerase
00:40:00.19 deletion response, the TDR. Now we found a set of
00:40:05.02 genes uniquely upregulated in response to the deletion of
00:40:09.17 telomerase RNA, and we called that the "telomerase
00:40:12.04 deletion signature." It was a particular group that
00:40:15.08 behaved as a group only when you deleted the
00:40:17.18 telomerase RNA, so that would be a group of those that
00:40:20.19 showed up as red, because they were upregulated. And
00:40:24.01 that hadn't been seen by any other manipulation. So the
00:40:27.16 cells indeed were sensing that they were losing
00:40:30.16 telomerase, and they responded in a particular way, by
00:40:34.08 changing their physiology, which we read out as a
00:40:36.21 change in gene expression profile. Right at the stage of
00:40:41.27 senescence, when I showed you that there were a lot of
00:40:44.13 genes that particularly were turned up or down in their
00:40:48.03 activity, there was a particular known DNA damage
00:40:54.01 response profile that was very prominent. That actually
00:40:57.25 makes a lot of sense. If you remember, I told you that,
00:41:00.24 when telomeres become short, they will become prone to
00:41:04.05 fusions, and now when they fuse, then chromosomes will
00:41:07.21 get ripped apart as they try to go through the ensuing cell
00:41:11.23 divisions, because the chromosomes with fused telomeres
00:41:14.26 will now have two centromeres that will pull apart in
00:41:17.15 mitosis, breaking the chromosomes at random positions,
00:41:21.09 and causing essentially genomic havoc, which can
00:41:23.27 quickly lead to cell death if the cells try to keep dividing.
00:41:28.09 And there's a damage response that takes place when
00:41:31.23 DNA breaks appear, and this is what we see, so this
00:41:34.29 makes very good sense. At senescence, we see a
00:41:37.14 damage response. What was very intriguing was that we
00:41:41.03 also saw others things. We saw what's called an
00:41:42.25 "environmental stress" cellular response. This is a
00:41:46.17 common set of genes that are upregulated and
00:41:49.00 downregulated in response to various insults, chemical
00:41:52.28 insults of various kinds, but it's a common set of genes, so
00:41:57.09 this is called the environmental stress response, and we
00:41:59.19 saw that response occurring, particularly at senescence.
00:42:05.05 And there was a change to an aerobic metabolism
00:42:08.15 program, and indeed we found that the number of
00:42:10.05 mitochondria at senescence went up enormously. Very
00:42:13.11 intriguing observations, not expected. Now, the survivors
00:42:20.02 were fascinating because, even though I showed you
00:42:22.26 they appear to grow quite well, they had a gene
00:42:26.15 expression transcriptional profile which was distinct from
00:42:29.24 the wild-type cells. And what persisted was a subset of
00:42:34.27 the environmental stress response genes that stayed on,
00:42:39.27 so what's interesting is that those cells, which appear to
00:42:42.10 be growing well on the surface, "stoics," they're really
00:42:45.27 hurting; their physiology is different, and they sense
00:42:49.25 themselves as being under what they sense as a cellular
00:42:53.23 stress. So, you can have cells that grow well without
00:42:58.20 telomerase, but if you look at the cells, these cells are
00:43:02.05 behaving as though under an environmental stress, and
00:43:05.25 so they indicate by their gene expression profiles a cellular
00:43:09.21 stress response. So in fact, cells do grow better with
00:43:15.04 telomerase than without it, even though superficially they
00:43:18.20 look similar, cells growing without telomerase maintaining
00:43:22.00 their telomeres via this recombination type of mechanism,
00:43:25.24 which is presumably somewhat more haphazard, is putting
00:43:28.27 a continuous stress on the cells, even though they've
00:43:32.04 adapted to it by a stress response. So, what we learned
00:43:38.09 then is that yeast lacking telomerase, using recombination
00:43:42.09 to maintain telomeres, do grow well, but they're under
00:43:45.10 continuing cellular stress, and this is what these gene
00:43:48.29 expression profile analyses showed us. So, let's go back
00:43:55.28 to telomerase and consider again what it's doing. If you
00:44:00.19 have plenty of telomerase, as indeed yeast cells normally
00:44:05.00 do, unless we genetically or in other ways ablate the
00:44:08.13 action of telomerase... if you have plenty of telomerase,
00:44:11.25 then there's kind of a homeostasis of the telomeres. The
00:44:15.21 telomeres stay within a certain range of lengths, and
00:44:19.06 remember I said at the very beginning of this lecture that
00:44:22.23 telomeric repeats are different in different molecules of a
00:44:27.02 population of cells, in different DNA molecules. And in
00:44:31.03 fact, they distribute around an upper and a lower limit.
00:44:35.27 And telomerase is one of the things that's crucial for
00:44:39.06 keeping them within this limit, and if you don't have
00:44:41.25 telomerase, then because of the DNA replication
00:44:46.05 problems, they gradually fall below. But they don't get too
00:44:49.25 long either, and a great many factors limit the action of
00:44:54.01 telomerase on telomeres as well, so they don't get too
00:44:57.05 long, and telomerase is also regulated on telomeric ends,
00:45:03.22 so that as the telomeres get shorter, then telomerase has
00:45:07.07 a higher probability of acting on the telomeres. This is a
00:45:09.25 complex system, we call it a homeostasis type of system,
00:45:14.27 and it balances out the lengthening and shortening
00:45:17.23 processes, so that the net result is that the telomeres stay
00:45:21.02 some average length within limits, and therefore the cells
00:45:24.21 keep dividing. Now, everything I've said implied that, if
00:45:32.22 you took away telomerase, there would be quite a delay
00:45:37.11 before any response was seen, and indeed, at a gross
00:45:42.09 level that is true. But if one scrutinizes the cells very
00:45:46.21 carefully, one sees that actually, things are a little bit
00:45:50.12 different. So an experiment in yeast was done, which was
00:45:54.26 to remove telomerase, as I showed you, and if you look at
00:45:58.00 the cells, I showed you growing on the plate, lots of
00:46:01.10 colonies, right away things look pretty okay. But if one
00:46:07.12 used very sensitive, quantitative molecular probes to look
00:46:12.05 at the telomeres in those cells, still with their long
00:46:16.22 telomeres, one found that there were occasional... every
00:46:20.22 few thousand cells or so had very short telomeres, and
00:46:24.28 they would actually fuse with a DNA break that was
00:46:28.13 induced to occur in those cells, something a telomere
00:46:33.05 never should do if it's a properly kept, functional telomere.
00:46:37.24 So, one in a few thousand cells had dysfunctional
00:46:42.17 telomeres, as manifested by the fact that they underwent
00:46:46.09 these normally forbidden fusion events. So immediately,
00:46:52.23 there was a response, even if one made the telomeres all
00:46:55.25 longer and then took away telomerase, one still saw this,
00:46:59.16 even when the bulk telomeres were long, one could see
00:47:02.16 this kind of aberrant dysfunctional telomeres immediately
00:47:09.03 after loss of telomerase. So this is one piece of evidence
00:47:14.20 that telomerase actually is protecting the ends of
00:47:18.11 telomeres, even when they're plenty long enough,
00:47:20.25 telomerase itself seems to protect and sit at the ends of
00:47:24.21 telomeres, is one interpretation, and protects them from
00:47:29.14 catastrophic shortening and fusioning to a double-strand
00:47:32.10 break, even when telomeres are long. This doesn't
00:47:36.28 happen in a high number of the cells, but it happens
00:47:41.10 measurably, and so this protective function of telomerase
00:47:46.18 is important. So I've introduced you to telomerase, I've
00:47:52.15 shown you how it's important for the long-term growth of
00:47:54.21 cells because it is necessary for continuous replenishment
00:47:58.28 of telomeres, and I've introduced you to the first piece of
00:48:02.18 experimental evidence that telomerase has a protective
00:48:06.01 function in cells, even when telomeres are long. So in the
00:48:11.06 next lecture, we'll talk about telomerase and more of its
00:48:16.28 protective functions in different cellular settings.
00:48:22.26

Part 2: Telomeres and Telomerase in Human Stem Cells and in Cancer

00:00:07.08 Welcome to the second part of this three-part lecture
00:00:11.04 series that I'll be giving on telomeres and telomerase. In
00:00:14.21 part two, I'm going to discuss telomeres and telomerase in
00:00:18.13 human cells, and particularly I'm going to emphasize the
00:00:22.16 setting of cancer cells. Now you may recall from the first
00:00:27.04 lecture that the function of telomerase is to maintain the
00:00:31.23 telomeres and prevent them from shortening as cells
00:00:36.04 divide, because telomere shortening would otherwise
00:00:39.00 occur in the absence of telomerase, to compensate for
00:00:42.11 the shortening processes. And so, maintaining telomeres
00:00:46.14 allows the cells to keep on dividing. In human cells, we
00:00:54.09 can also see though that telomerase itself is protecting
00:00:58.22 telomeres, and so I want to show you one kind of
00:01:01.04 experiment for that. And the conclusion is going to be that
00:01:08.01 it's not just the bulk telomere length that matters, but the
00:01:11.18 presence of telomerase can determine whether a
00:01:15.02 telomere is seen by the cell as sufficiently long or not. The
00:01:21.26 experiment I'll show you was done in cultured human
00:01:25.09 cells. These were not cancer cells, and these were cells
00:01:28.22 that have normally extremely, extremely low levels of
00:01:31.22 telomerase, effectively, for our purposes, essentially no
00:01:35.16 measurable telomerase activity, and certainly their
00:01:37.25 telomeres are not maintained. Now I've shown you in very
00:01:40.26 simple diagrammatic form human telomerase, so this
00:01:44.20 would be the template sequence of the human
00:01:47.21 telomerase RNA, and this is the overhanging G-rich
00:01:53.17 strand. It's actually usually longer than this, but I've just
00:01:56.26 shown it as a simple diagrammatic form here. And this of
00:02:00.23 course is the duplex telomeric DNA that will be consisting
00:02:06.06 of hundreds or thousands of telomeric repeats, as you go
00:02:09.02 in toward the chromosome interior. And just as I showed
00:02:14.00 you for Tetrahymena telomerase, the template region is
00:02:17.28 copied, and so the DNA... for example, a DNA with three
00:02:22.15 Gs, two Ts, and an A, would sit down here on the
00:02:25.13 template, and then nucleotides would added, extending
00:02:28.13 along the template, thereby lengthening the DNA in this
00:02:34.01 reverse transcriptase reaction that telomerase carries out.
00:02:39.27 So what was done was to compare cells in which
00:02:43.29 telomerase was being expressed or not being expressed,
00:02:49.15 and look at the growth of the cells and the telomere
00:02:52.21 length. Now the protein TERT is the core protein that has
00:02:57.11 this enzymatic activity: the reverse transcriptase activity,
00:03:01.28 and probably other activities, as I will allude to. And so, in
00:03:06.29 these experiments, a particular mutation was made on the
00:03:11.07 TERT protein, it happened to be a very small change of a
00:03:13.27 few amino acids added to the very C-terminus, and what
00:03:18.29 this does is it doesn't affect the enzymatic activity, but it
00:03:22.25 does affect the ability of this enzyme to elongate
00:03:27.10 telomeres in cells, and it's called a hypomorph because
00:03:32.21 that refers to the fact that it has an insufficient function,
00:03:37.26 but it showed something useful. Now here's the
00:03:40.28 experiment, and I'm going to walk you through this rather
00:03:43.28 complex-looking slide. First of all, I want to show you a
00:03:47.14 growth curve of human fibroblasts in culture. What
00:03:51.10 normally happens is, if we look at the... this is cumulative
00:03:55.03 dilutions, which is just an operational term telling you how
00:03:57.29 many cell divisions are going on, and this is the number of
00:04:00.29 days. So if you culture human fibroblasts, normally what
00:04:04.08 you find is that the cells will continue to multiply for a
00:04:08.04 while, and then they'll cease multiplying any further, so the
00:04:11.19 curve flattens out, so you see they've undergone
00:04:14.11 something like 50 or so divisions. And if you put in a
00:04:19.02 control vector, which would be important, you get the
00:04:22.07 same curve. But if you put in the test vector, which
00:04:26.09 actually is expressing this form of the human telomerase
00:04:31.07 core protein TERT, because the fibroblast cells naturally
00:04:35.29 have enough telomerase RNA and the other components
00:04:38.14 of telomerase, all they're missing is the TERT, you just
00:04:40.27 have to add this in, and now you can restore telomerase
00:04:44.08 activity. But what's very interesting is the telomeres and
00:04:47.26 the cell growth. What you can see is, first of all, the cell
00:04:51.19 growth has been greatly extended. We've now made
00:04:54.15 these cells very much elongated in their lifespan
00:04:59.03 compared with the controls or the parental cells. Now let's
00:05:03.11 look at the telomeres. If we look at the telomeres in these
00:05:07.27 cells, the controls, we find that they're gradually becoming
00:05:12.07 somewhat shorter, and at this point here, they've pretty
00:05:15.13 much ceased to divide, so we're out here, they pretty
00:05:18.20 much cease dividing. So the telomeres on average are
00:05:21.24 about this long, and the cells have picked up the signal,
00:05:24.27 they've said the telomere length is shorter here than here,
00:05:28.01 and they've picked up the signal, and they've said we're
00:05:29.16 not going to divide any further. Now we've added the
00:05:32.27 hTERT. Now actually what happened was that, I told you
00:05:35.27 the cells continue to grow a long, long time. The
00:05:39.10 telomeres shorten, and then they steady out at some
00:05:43.11 much shorter length, so through here and through here,
00:05:46.09 they're actually growing quite well, but they're going with
00:05:48.19 much shorter telomeres. So in other words, these cells
00:05:55.13 can keep dividing for a long time with very short
00:05:58.09 telomeres, and the difference was they had telomerase
00:06:02.03 being expressed. So we used a trick just to separate out
00:06:06.05 the telomere lengthening property of telomerase from its
00:06:10.02 ability to stabilize telomeres, and we saw that in fact if you
00:06:13.27 have this telomerase present throughout, even though
00:06:17.04 the telomeres maintain short, they are perfectly stable,
00:06:21.09 and the cells can keep dividing. So this is the second
00:06:25.24 piece of evidence, I showed you the first piece of
00:06:29.00 evidence for you in yeast systems in the first part of this
00:06:33.05 lecture series, and now this is the second piece of
00:06:35.20 evidence that telomerase is having a protective function,
00:06:39.10 in this case, it's in human cells, and it's stabilizing
00:06:42.12 telomeres that otherwise would've been too short in its
00:06:45.08 absence. This is not unique to human cells, it's been seen
00:06:48.27 in yeast systems as well experimentally. So I just talked to
00:06:54.02 you about normal cells, and I told you that, in those
00:06:58.11 human fibroblasts grown in culture, there's very little
00:07:01.22 telomerase. So, now let's talk about where do we normally
00:07:10.09 find telomerase in human cells? Well, if you look in cells,
00:07:20.14 you find that it actually is on at times when the cells are
00:07:23.18 greatly proliferating during fetal development, and it does
00:07:26.25 remain active in certain proliferative cells. It's also found
00:07:31.15 active in stem cells, in cells that are activated to
00:07:35.24 proliferate, such as lymphocytes proliferating under the
00:07:41.20 response to, for example, a pathogen. One finds stem
00:07:47.01 cells, for example, in hair follicles; those have telomerase.
00:07:51.07 So one does find telomerase in cells that are stem cells
00:07:54.27 and various sorts of cells that are induced to proliferate.
00:08:02.17 And in fact in most other cells, one can find telomerase,
00:08:06.23 but it's in very low levels. So initially people thought there
00:08:10.03 was no telomerase in normal epithelial cells or fibroblasts
00:08:13.14 or endothelial cells, but closer scrutiny showed that in fact
00:08:17.20 there were real, definitely low and very downregulated,
00:08:21.16 but real levels of telomerase. And in the third part of this
00:08:29.02 three-lecture series, I will talk more about the telomerase
00:08:34.10 in the normal cells of people and tell you about some in
00:08:40.05 vivo studies that have been done. Now, cancer cells.
00:08:45.24 Cancer cells are infamous for their ability to keep on
00:08:50.11 multiplying. Now, they can do this for a variety of different
00:08:54.21 reasons. They lose their system of checks and balances
00:08:58.11 that prevent them from overmultiplying, and that's
00:09:01.07 because of a lot of genetic and epigenetic changes that
00:09:04.11 have taken place in their progression to become a
00:09:08.08 malignant cancer cell. Now, they are immortal cells, and
00:09:13.17 indeed, as you might expect, telomerase is on in these
00:09:16.19 cells, and in fact it's very high in the vast majority of
00:09:20.19 human cancers, particularly as they've got to the invasive
00:09:23.24 stages. And that makes a lot of sense based on what I
00:09:28.16 just told you, because if the cells are to keep on
00:09:31.01 multiplying, then they have to keep on replenishing their
00:09:34.01 telomeres. Now I'll you some more recent results in the
00:09:38.27 last few years, which also suggest telomerase may be
00:09:42.16 doing other things in human cancer cells. It is certainly
00:09:46.14 maintaining the telomeres, and that is important, but there
00:09:49.28 may be other functions as well. So I'll tell you some newer
00:09:52.21 findings about that. So, let's just recapitulate what I said
00:09:58.06 about where we find telomerase in humans. So it keeps
00:10:01.19 telomeres elongating and replenished, and therefore cells
00:10:04.26 can keep dividing in stem cells and, of course, I didn't
00:10:10.00 mention germ cells. Now of course we wouldn't be here if
00:10:13.05 we didn't have maintenance of telomeres from generation
00:10:16.03 to generation. Telomerase indeed is active in germ cells,
00:10:20.16 as well as stem cells of various kinds. As I said, it's
00:10:23.25 detectable in many normal cell types, and highly active in
00:10:27.03 the great majority of human cancers. And by the way, in
00:10:32.14 some of those human cancers in which you don't find the
00:10:35.09 high telomerase, one actually often finds this ALT
00:10:39.03 mechanism, but it's only a particular subset of cancers in
00:10:43.19 which one finds ALT being a prominent means of
00:10:47.25 maintaining telomeres. The great majority of the common
00:10:50.26 human tumors have highly active telomerase. So,
00:10:56.15 telomerase is highly active in human tumors. So one could
00:11:02.24 imagine inhibiting telomerase, and this might be a good
00:11:07.18 target for trying to inhibit the growth of cancer cells, if you
00:11:12.16 could inhibit telomerase in cancer cells. And so in
00:11:17.01 investigating this, some interesting things emerged. So
00:11:21.25 let's just think about what is expected to happen if you
00:11:24.09 don't have telomerase in the cancer cells. Well, as you
00:11:27.27 know, cells multiply, and their telomeres will progressively
00:11:31.05 become shorter and shorter if there isn't telomerase to
00:11:33.27 counteract that shortening, and so eventually when the
00:11:37.18 telomeres get too short, the cells would eventually cease
00:11:40.14 to divide, and the cells respond to those short telomeres
00:11:45.25 by either what's call the "senescence response," in which
00:11:49.02 the cells simply won't replicate their DNA anymore, or a
00:11:53.14 cell death response that can include an apoptotic
00:11:56.25 response, which involves an active cell suicide program
00:12:01.04 that can be induced by dysfunctional telomeres. Whether
00:12:05.15 it's senescence or cell death does depend upon the cell
00:12:09.11 type. So, the simple prediction, diagrammatically, would
00:12:14.03 be, if you didn't have telomerase, the cells now would
00:12:18.11 have shorter and shorter telomeres, and after some
00:12:20.29 number of divisions, eventually there would be cell death.
00:12:27.18 Without telomerase, it's been observed experimentally,
00:12:30.06 typically human cells lose their telomeric DNA at this kind
00:12:34.08 of rate. I've put 150-200 base pairs per cell division; that's
00:12:39.02 seen in a number of cultured cells and also in some
00:12:45.00 cancer cells in which telomerase has been inhibited.
00:12:48.21 Now, I told you that human telomeres are typically made
00:12:53.13 of hundreds, even thousands, of copies of telomeric
00:12:59.26 repeats, they're thousands of base pairs long, so at this
00:13:02.27 rate, it's going to take quite a lot of cell divisions before
00:13:06.03 the telomere gets short enough that the cells will have this
00:13:09.25 response. So that in fact is the prediction, and if you
00:13:15.19 inhibit the telomerase enzyme using, for example, a small
00:13:19.17 molecule inhibitor that inhibits the catalytic function of
00:13:24.00 telomerase that prevents it from carrying out that DNA
00:13:28.06 polymerase reaction by the reverse transcriptase
00:13:31.22 mechanism, if you inhibit telomerase in such a way,
00:13:35.22 indeed this is the observation. There's a gradual
00:13:38.03 shortening of telomeres, and eventually the cells cease to
00:13:41.01 divide. So this is seen in cancer cells in culture treated
00:13:44.20 with such an inhibitor. Now I've put a noted point up here:
00:13:50.29 but you're still keeping the telomerase ribonucleoprotein
00:13:55.15 (RNP) level high. You're not depleting the cells of the
00:14:00.16 enzyme, you are simply rendering that enzyme inactive.
00:14:07.25 And I make that distinction because of the next results I
00:14:10.29 want to tell you about. So, what was observed was quite
00:14:17.17 surprising. If one depleted telomerase, and the particular
00:14:22.04 way to knock the telomerase RNA down, then one found
00:14:26.07 there was a very rapid effect on human cancer cells. One
00:14:30.22 didn't see the long delay ensuing before the effect was
00:14:37.03 observed. So, now I'll tell you about those experiments.
00:14:42.29 So, reminding you again, here's human telomerase, it's
00:14:45.12 copying its RNA template, and of course the enzyme has
00:14:50.14 the TERT protein and the telomerase RNA, and it's the
00:14:54.10 RNA that is going to be depleted in these experiments.
00:14:59.15 How's the RNA depleted? A now commonly used
00:15:02.28 technique, which is called a knockdown using RNA
00:15:06.24 interference. Now the particular technique was to express
00:15:11.22 from a lentiviral vector, which you can introduce efficiently
00:15:16.03 into cells, a particular sequence which forms a double-
00:15:21.10 stranded RNA, and I've just shown the corresponding
00:15:23.27 DNA sequence here, and that double-stranded RNA can
00:15:27.23 interact with a cellular RNA that matches its sequence,
00:15:32.08 and eventually cause the breakdown of that RNA
00:15:37.00 through a complicated process known as RNA
00:15:39.19 interference. And so, siRNA refers to "short interfering
00:15:44.24 RNA," because such a short interfering RNA, which
00:15:49.00 involves introducing two strands of RNA complementary
00:15:53.24 to the target RNA, that is what causes the breakdown to
00:15:58.05 occur. Now when we did this experiment, so here's a
00:16:01.22 control where we're looking at a reference amount, here's
00:16:04.18 the telomerase RNA, one found that in fact the method of
00:16:09.16 introducing such a short interfering RNA by this kind of
00:16:14.06 construct here (the details don't matter), one could in fact
00:16:18.02 quite efficiently knock down the telomerase RNA in
00:16:22.17 human cancer cells grown in culture, for example, in
00:16:25.26 these breast cancer cells grown in culture. And so one
00:16:30.15 could knock it down almost down to about 10-12% of the
00:16:36.16 original level that one sees in the controls. So what
00:16:41.27 happens then? Now, if what I had told you was the case,
00:16:47.05 that we knock down the telomerase, then we would
00:16:49.20 expect to have to wait for a long time for the telomeres to
00:16:53.20 get short enough, if that were the only thing going on,
00:16:57.05 then we would have to wait before we saw any effect.
00:16:59.10 But right away, there was an effect on the growth of the
00:17:02.14 cells. Now just to put this into perspective here, I'll just
00:17:07.25 walk you through a couple of graphs. Here's the controls
00:17:10.00 of two kinds, and this is the number of cells here. And so
00:17:14.10 what you can see is that the cell number goes up and up
00:17:17.04 and up, as you might expect. The cells are dividing once
00:17:22.11 every day or two, and so you can see, after about four or
00:17:25.13 five days after the introduction of the construct that
00:17:29.29 knocked the telomerase RNA level down, that the cells
00:17:34.13 that received this are quite quickly growing more slowly
00:17:40.04 than the controls. So this must be occurring within a
00:17:43.07 couple of cell divisions. And here are some more controls
00:17:47.07 in which, here's the empty vector, here is the short
00:17:52.16 interfering RNA targeting telomerase RNA, and here's a
00:17:56.07 control version of that, in which it now no longer can
00:18:00.12 target the telomerase RNA, but everything else is the
00:18:03.08 same, and as you can see it behaves like the controls.
00:18:06.03 And so a great many control experiments were done to
00:18:09.29 show that this was a specific effect specific to knocking
00:18:13.27 down the telomerase RNA. This was done on bulk,
00:18:20.27 unselected cell populations. The cells were a melanoma
00:18:27.02 cell line that normally has very long telomeres. The bulk,
00:18:32.01 unselected populations is significant because what it
00:18:36.24 meant was that one could put the siRNA, the agent that
00:18:41.14 knocks the telomerase RNA down, into the cells at day
00:18:45.21 zero, and without even any selection, one could get
00:18:51.10 something like 80-90% of the cells that received this
00:18:54.15 construct, and in fact the ones that did grow out were
00:18:57.29 that low percentage that didn't receive the construct. So
00:19:01.21 in fact the effect is even stronger than what these curves
00:19:05.17 would indicate, because these ones that are growing out
00:19:09.08 are largely the ones that just didn't receive the construct.
00:19:17.07 Now, one of the things that is very important in human
00:19:21.16 cells to respond to DNA damage such as certain kinds of
00:19:26.09 telomeric DNA damage is the gene p53. Interestingly,
00:19:33.01 these effects did not require p53. Here is a pair of cell
00:19:37.19 lines that are otherwise isogenic. This is a colon cancer
00:19:41.17 cell line called HCT116, in these cells the p53 is wild-type,
00:19:46.26 here's the response to knocking down telomerase RNA.
00:19:50.26 In this otherwise isogenic cell line lacking p53 but
00:19:56.00 otherwise the same, this response is quantitatively the
00:20:00.00 same. So this is a different response from a classic DNA
00:20:05.14 damage response, and in fact we did not see DNA
00:20:08.20 damage response genes being induced here. We looked
00:20:14.03 at the telomeres by some molecular probing mechanisms
00:20:21.29 that allow one to see if the telomeres are uncapped, and
00:20:25.00 in fact they were not uncapped either. So we see, when
00:20:30.09 we knock telomerase RNA down, we rapidly see an
00:20:33.04 inhibition of cancer cell growth, these are cells that
00:20:36.06 normally have very high telomerase. p53 is not required
00:20:39.26 for this, suggesting it's not a classic DNA damage
00:20:42.18 response. And the telomeres, as far as we can see, are
00:20:46.01 not uncapped, and there's not DNA damage response.
00:20:50.01 Indeed, the telomeres hadn't had time to shorten
00:20:52.14 perceptively at all during this short timeframe. So the rapid
00:20:58.00 knockdown doesn't uncap the telomeres. That's not the
00:21:01.06 cause of the growth inhibition of these human cancer
00:21:04.25 cells. But interesting things happen very quickly to these
00:21:10.20 cells when you knock down telomerase RNA. I'm going to
00:21:15.11 show you this one experiment in which, in this experiment,
00:21:22.01 melanoma cells were grown in culture, and the RNA level
00:21:25.10 was knocked down by a completely different mechanism,
00:21:28.21 it's called a "ribozyme," and the purpose of it is to cleave
00:21:36.09 the telomerase RNA, causing it to break down, and so it
00:21:39.22 has exactly the same effect that I showed you for the
00:21:43.03 RNA interference: you knock down the telomerase RNA
00:21:46.10 level. A very interesting thing was seen. These are cells
00:21:51.14 that are growing in culture, and these are three flasks of
00:21:54.10 cells that either are control cells that just got the empty
00:21:57.08 vector. The cells are all growing on the bottom of the
00:21:59.11 flask, you can't see it. We're just looking at the medium,
00:22:03.02 the broth, the liquid medium in which the cells are
00:22:05.22 growing, and it's a nice, pinkish color here in the controls.
00:22:09.26 But here, in two separate versions of the cell lines that
00:22:15.26 received the construct that knocked the telomerase RNA
00:22:19.07 levels down, you can see that the medium has turned a
00:22:22.20 dark color. These were melanoma cells, so it was of
00:22:27.06 course very easy to wonder if indeed these cells were
00:22:31.17 now producing the pigment melanin in high amounts in
00:22:35.15 this and this but not in the control cells. And indeed,
00:22:40.13 analyses showed that that was exactly what was
00:22:42.22 happening. And in fact, a lot of interesting things
00:22:46.11 happened. These cells became more like their normal
00:22:50.21 counterpart cells. It was as though they were more
00:22:53.09 differentiated; they looked more dendritic in form, as
00:22:57.15 though they had become less cancerous. And indeed
00:23:00.18 their gene expression profiles had changed. So, knocking
00:23:06.01 down telomerase RNA also made these cells less
00:23:11.23 invasive. The more differentiated the cancers are, very
00:23:15.17 typically, the less invasive they are, and in fact, in
00:23:20.21 preclinical mouse model systems used, in fact metastasis
00:23:24.13 was knocked down. And that was seen whether one
00:23:27.15 knocked telomerase RNA down with the ribozyme, or with
00:23:31.13 the RNA interference mechanism, but in melanoma
00:23:36.17 models for cancer, using the experimental mouse system
00:23:41.01 in the laboratory, it was found that the metastasis is
00:23:48.10 decreased when the telomerase RNA is depleted. So
00:23:51.14 knocking telomerase RNA levels down changed the
00:23:55.01 nature of the cells, and it changed the nature of the cells
00:23:58.12 well before the telomeres became uncapped. So, as I
00:24:05.15 said, when you have plenty of telomerase, you expect
00:24:09.11 cells to be able to multiple indefinitely, and you don't
00:24:12.24 expect any effects to occur, of removing telomerase, until
00:24:18.28 a long time has elapsed. So, what we see by this abrupt
00:24:26.14 knockdown of telomerase level, by knocking down the
00:24:29.14 telomerase RNA, is we see rapid inhibition of cell growth,
00:24:33.04 p53 was not required, there was no DNA damage
00:24:37.11 response or telomere uncapping, and in fact metastasis is
00:24:41.11 reduced. Metastasis is reduced, what is going on? I briefly
00:24:50.29 mentioned, we looked at the gene expression profiles in
00:24:54.19 these cells, and we found that in fact that rapid
00:25:00.00 knockdown of telomerase RNA and that rapid slowing of
00:25:05.00 the cell growth is accompanied by and presumably
00:25:08.15 caused by cell cycle and tumor progression genes being
00:25:13.00 downregulated. Glucose metabolism is downregulated;
00:25:18.13 cancer cells typically have high rates of glucose
00:25:21.25 metabolism. That becomes downregulated in cells that
00:25:25.12 have less telomerase RNA. And the cells appear more
00:25:30.04 differentiated, making one wonder if there's a cell
00:25:33.06 differentiation program that's induced in these cells.
00:25:38.12 Unexpected changes, many open questions remain, but
00:25:43.00 these kinds of experiments have led to these
00:25:45.21 observations here that have two kinds of implications:
00:25:50.28 One is the scientific implication that telomerase has other
00:25:55.01 functions that are not solely mediated through its adding
00:25:59.27 telomeric DNA to the ends of the chromosomes, and the
00:26:03.25 other implication is that this makes telomerase an
00:26:08.16 interesting target for potential anticancer therapies. And
00:26:19.13 the take-home message that I think is most impressive
00:26:23.09 and unexpected was, there seems to be good reason to
00:26:27.15 think that high telomerase levels are promoting an
00:26:30.13 undifferentiated, "stem cell-like" phenotype, a very
00:26:34.16 unexpected observation. And in fact, now in other
00:26:38.09 systems, I won't have time in this lecture to go through it,
00:26:42.02 but there's now evidence that this is really the case, in
00:26:47.28 even noncancerous cells in certain model organisms
00:26:52.28 where this has been studied.
00:26:56.02

Part 3: Stress, Telomeres and Telomerase in Humans

00:00:03.18 Welcome to part three of the series of three lectures on
00:00:07.29 telomeres and telomerase. This third part is going to be on
00:00:12.01 stress, which I will define, and telomeres and telomerase
00:00:18.01 in humans. In the previous two lectures, you'll remember
00:00:23.00 that we talked about the fact that, if you have plenty of
00:00:25.20 telomerase, then homeostasis in the sense of telomere
00:00:29.24 length maintenance can be maintained. There's a
00:00:32.24 balance of lengthening and shortening processes on
00:00:36.11 telomeres, so that an average length of telomeres is
00:00:39.24 maintained, and the cells can keep dividing, if there's
00:00:42.20 enough telomerase and other conditions are met,
00:00:45.04 essentially indefinitely. So now I want to put this kind of
00:00:52.14 cellular and molecular information that we've been gaining
00:00:57.10 about telomeres and telomerase into perspective with
00:01:00.24 respect to humans and a question that is greatly of
00:01:05.24 interest to many people, and that is the question of, how
00:01:08.13 do we age? What underlies this familiar kind of
00:01:15.01 progression shown in this series of pictures here? The first
00:01:19.20 thing I'd like to emphasize is that aging is a multifaceted
00:01:23.29 process. I think there's plenty of reason to think that that's
00:01:28.19 the case, and there won't perhaps be a single answer. So
00:01:32.29 I will really focus on one particular aspect of it. And even
00:01:38.08 at the level of whole humans, we can think of multiple
00:01:42.28 facets of the process of aging, and one of them is the
00:01:47.00 observation that there's increased susceptibility to certain
00:01:50.11 kinds of diseases. And we can wonder about this, and
00:01:56.11 wonder how much of this is environmental (life factors,
00:02:00.14 stochastic), and how much of this is genetic? And of
00:02:05.05 course, no doubt there'll be much interaction between
00:02:09.12 those two. I'm going to talk about some work studying
00:02:15.19 telomere maintenance in humans and show an interesting
00:02:23.00 set of findings that suggest that the environmental and life
00:02:27.16 factors aspects can certainly play a role. And that is not to
00:02:33.05 say genetics is not important, and I'll show you a sort of
00:02:36.07 situation where it's clear genetics is important as well. So
00:02:42.20 if we think about this susceptibility to diseases and
00:02:46.10 mortality, one can wonder about the genetic and the
00:02:50.27 nongenetic aspects, and I'm going to show you a graph
00:02:53.26 that came from Gavrilova and Gavrilov just to give you an
00:02:57.11 example of the fact that the genetic and the nongenetic
00:03:01.17 components of aging are not just monotonic across the
00:03:05.13 decades of human adulthood. So this was a study of over
00:03:09.29 5000 daughters, of well-to-do daughters of well-to-do
00:03:14.01 families in Europe, and which there was good
00:03:16.09 genealogical information as to the fathers' lifespan and
00:03:19.27 the mothers' lifespan and the daughters' lifespan. And so
00:03:23.03 with this database of over 5000 daughters with such
00:03:26.03 complete information, and all of whom were well-to-do, the
00:03:31.27 question was asked: Of the daughters who lived to be 30
00:03:34.08 years and older, how did their lifespan relate to either their
00:03:39.06 fathers' lifespan, which is shown here, as I'll explain, or
00:03:43.07 the mothers' lifespan, and I'll just tell you about that. But it
00:03:46.11 was very similar to what I've shown you here for the
00:03:48.07 pattern of lifespan. And the relationship was this: If you
00:03:51.21 looked at daughters who lived to be a certain age and
00:03:56.08 asked what their life expectancy was compared with the
00:04:00.13 rest of the daughters who were born in those same years,
00:04:04.02 and compared with how old their fathers grew to be
00:04:07.27 before they died, in other words, what was their fathers'
00:04:09.29 lifespan, for people who had fathers who lived to be 75
00:04:15.07 years and older, there was really quite a strong
00:04:18.10 relationship between the greater the father's lifespan was,
00:04:23.24 the greater the chance that the daughter would live
00:04:26.22 longer than expected. And so this function, which is
00:04:30.15 called a residual, basically increases with parental
00:04:34.28 lifespan, at least just for the father, 75 years and older,
00:04:40.21 whose life expectancy was 75 years and older, and that
00:04:46.09 was the relationship expected if genetics is an important
00:04:52.22 contributor to the daughters' lifespan. So if the father lived
00:04:58.22 longer than 75 years, then the daughter's lifespan was
00:05:04.16 related to it in a way that said genetics was important. But
00:05:08.25 what about all the fathers who didn't live to be 75, who
00:05:11.17 lived to be less? There, the relationship was very random,
00:05:19.05 so in other words, it made little difference whether your
00:05:23.16 father had a lifespan of 40, or your father had a lifespan of
00:05:26.03 70 years, to what your lifespan, the daughter's lifespan,
00:05:31.14 was. So in other words, mathematically, this function
00:05:38.03 around zero, that's expected if actually inheritance is
00:05:41.16 unimportant. Very similar plot was seen when it was the
00:05:46.15 maternal lifespan instead of the paternal lifespan that was
00:05:51.04 plotted out. So in other words, for this great majority of the
00:05:56.15 daughters, over 70% of the daughters fell into this
00:06:03.11 category, in fact things that are not genetic (we could call
00:06:08.18 them environmental or stochastic, life factors...
00:06:11.09 nongenetic factors) were clearly more overwhelming
00:06:15.07 quantitatively. So the genetic component was much more
00:06:20.12 important to fathers with a long lifespan than it was for the
00:06:24.25 fathers who had this wide range of somewhat shorter
00:06:30.02 lifespans. It just says that the relationships can be more
00:06:33.02 complicated. And so many summaries have been put
00:06:36.19 forward for this kind of observation, and I've added in
00:06:40.08 another one, and so that is elderly subjects demonstrating
00:06:43.11 exceptional longevity, for which I have just told you there
00:06:46.26 is certain kinds of genetic information that argues that this
00:06:51.28 is really a genetically based phenomenon, exceptional
00:06:56.18 longevity, such elderly subjects have generally been
00:07:00.23 spared the major age-related diseases, such as
00:07:03.20 cardiovascular disease, diabetes, and cancer. Now those
00:07:07.14 diseases are responsible for most deaths in the elderly,
00:07:11.27 and those are the ones for which one can find a lot of
00:07:14.10 nongenetic causes as well. So, cardiovascular disease,
00:07:22.23 diabetes, and cancer: Responsible for most deaths in the
00:07:26.24 elderly, and for which there's a significant nongenetic
00:07:31.11 component. So, how can one analyze this further, and
00:07:35.01 what's this got to do with telomeres? So that is what I'm
00:07:37.03 going to tell you about now, some relatively recent
00:07:40.01 research which connects these with telomere
00:07:43.14 maintenance now in humans. So to remind you, if
00:07:49.18 telomeres are replenished by continuous telomerase
00:07:53.15 action, then the cells can keep dividing. And in humans,
00:07:59.26 when one looks at the distribution of telomerase activity in
00:08:04.21 different cell types, one does find telomerase in stem cells
00:08:08.05 and germ cells, these are cells that are expected to be
00:08:11.20 essentially immortal cells. One does find it in human
00:08:18.24 tumors, but I'm not going to talk about that in this part of
00:08:23.08 this lecture series. But one finds telomerase at regulated
00:08:29.08 but real levels in a variety of other normal adult cell types
00:08:34.17 as well. So telomerase in the setting of normal cells is
00:08:39.08 found in stem cells, which are necessary for replenishing
00:08:43.28 tissues throughout the lifespan, such as immune system
00:08:48.22 stem cells, hair follicle stem cells are a one class, stem
00:08:53.13 cells in the gut, there are a lot of cells that replenish
00:08:57.01 tissues throughout life, and one finds telomerase in these.
00:09:01.10 But one also does find detectable levels of telomerase in
00:09:04.27 many, many normal adult nonstem cells as well. So, let's
00:09:12.12 just reiterate these expectations. If you have plenty of
00:09:15.19 telomerase, then homeostasis is going to be balanced.
00:09:18.02 Cells will keep dividing, so that would describe the
00:09:20.13 situation for stem cells, for example. Now, if you have just
00:09:26.00 a little bit of telomerase, then what's the situation going to
00:09:30.03 be like in that case? Well, if you had no telomerase at all,
00:09:35.16 we discussed in the first lecture what would happen.
00:09:38.18 There would be progressive shortening of the telomeric
00:09:41.10 DNA, and eventually when the telomeres became too
00:09:45.21 short, there'd be cellular senescence, and that is indeed
00:09:48.17 observed in some cells that have very, very little
00:09:52.00 telomerase when they grow in culture. But, the situation in
00:09:57.02 normal cells in the human body, not cells growing in
00:10:00.09 culture but the cells freshly analyzed from humans, as
00:10:04.15 fresh as you can get cells, and you look at them directly,
00:10:09.16 it's a more intermediate situation. So for example, what's
00:10:15.07 the prediction? If you had some telomerase, right, you're
00:10:17.12 going to have the shortening processes going on, but
00:10:20.01 each time the cells are dividing, perhaps there's some
00:10:21.21 telomerase, and so it's trying to keep it up, but eventually
00:10:25.15 it loses the battle. So, if you had some telomerase, the
00:10:28.19 prediction is that, since you haven't completely
00:10:32.00 compensated for the shortening processes, while
00:10:34.24 telomerase might be able to keep up, eventually the net
00:10:38.09 shortening will overcome the elongation ability of
00:10:42.03 telomerase, and senescence will eventually ensue later
00:10:46.05 than if you had no telomerase. And if you had some, a
00:10:49.14 little bit less but still some telomerase, then you'd get
00:10:52.16 there, to the point of senescence, but somewhat faster
00:10:55.15 than those cells that had a bit more telomerase. And so
00:10:59.12 it's these situations of this sort of gray zone that seem to
00:11:03.02 be the situation in normal human cells. Now this is
00:11:10.12 important because normal human cells will include certain
00:11:15.08 kinds of stem cells that are required to keep replenishing
00:11:20.19 throughout life, and then the proliferating cells that arise
00:11:23.28 from those stem cells that are required to regenerate
00:11:26.22 tissues throughout life, such as the immune system. So
00:11:31.00 how do we age? So we've learned a lot as we have
00:11:35.08 studied telomeres and telomerase in the laboratory in
00:11:38.28 model organisms that can now start to feed into this
00:11:43.28 question of, in humans and in people susceptible to
00:11:49.15 diseases of aging, how does this happen, how do we
00:11:55.20 age? And the converse can also be true. And let me
00:12:03.13 show you this very striking example: the clinic, the
00:12:07.18 bedside in this little diagram here. The clinic can be very
00:12:12.21 informative as to what might be going on in terms of
00:12:16.04 underlying biology. And such was the case with
00:12:20.23 telomerase. There's a rare, inherited condition in humans,
00:12:26.19 it's very rare, but it's now been seen in some dozens in
00:12:29.19 families. When we have billions of people on the planet,
00:12:32.13 then even a very rare disease can sporadically show up
00:12:35.22 in amounts that cumulatively add up to dozens of families.
00:12:39.00 This has been seen for a particular disease, it happens to
00:12:41.20 have the name "dyskeratosis congenita," which you don't
00:12:44.12 really have to remember, because it's not the most
00:12:47.04 clinically relevant aspect that's important here. What's
00:12:51.00 important is that such individuals die from progressive
00:12:56.16 bone marrow failure, and they suffer a premature death,
00:12:59.29 they don't make it to old age. And what's the defect? The
00:13:03.25 defect is that a copy of the telomerase RNA gene, and
00:13:08.10 the RNA is a single-copy gene found in one copy on a
00:13:13.04 particular chromosome. You get one copy of the
00:13:15.12 chromosome from your mother, one from your father, or
00:13:17.15 vice versa, so you normally have two copies of the
00:13:20.08 telomerase RNA gene, indicated as this little, yellow bar,
00:13:24.11 and on these chromosomes. So these are the two
00:13:27.00 chromosomes that have the telomerase RNA gene, one
00:13:30.07 that you got from your mother and one that you got from
00:13:31.24 your father. When one of those copies don't work, then
00:13:37.12 the primary clinical problem is progressive bone marrow
00:13:40.03 failure. This, as I said, causes premature death. Indeed it
00:13:44.21 can cause it early adulthood, sometimes even childhood,
00:13:49.25 to middle age. Such individuals have telomeres that are
00:13:52.27 way shorter than the normal range seen in healthy
00:13:56.06 individuals who do not have this inherited condition, or in
00:13:59.15 their family members who do not inherit this mutation. The
00:14:04.23 characteristic is that the immune system has become
00:14:07.12 exhausted, it looks probably as though stem cells, or the
00:14:10.23 cells that arise from stem cells in the immune system, just
00:14:13.28 lose the ability to proliferate all the way through a normal
00:14:18.00 life expectancy. As I said, the progressive bone marrow
00:14:21.11 failure, which is the primary cause of much of the death,
00:14:26.01 that is the problem, and the immune system then cannot
00:14:30.14 keep being replenished, which normally happens of
00:14:33.22 course from cells in the bone marrow. Interestingly, such
00:14:36.23 individuals are cancer-prone, they have very short
00:14:38.29 telomeres, and as I said in the first part of this three-
00:14:45.19 lecture series, telomerase can act to protect the ends of
00:14:50.29 chromosomes, and if we have deficient telomerase, then
00:14:54.12 that may be one reason why the chromosome ends are
00:14:57.24 dysfunctional, they're not protected, and secondly, the
00:15:02.24 telomeres are simply shorter, so they're also more prone to
00:15:07.02 becoming dysfunctional. So the important result, which
00:15:12.01 was found in 2001 by Vulliamy et al., was that mutating
00:15:17.27 one or other copy, from mother or father (it didn't make a
00:15:20.08 difference who had passed the gene down), but if one of
00:15:24.23 the copies of the telomerase RNA gene was mutated, as
00:15:29.17 is the case in the family members affected by this inherited
00:15:33.02 disease, even though the other gene copy is wild-type,
00:15:38.04 these are the ensuing consequences. The important
00:15:44.11 message is that to get through a healthy, full human
00:15:48.18 lifespan requires both the telomerase RNA alleles, that
00:15:54.07 means gene copies, to be functional. That means the
00:15:57.23 quantity of the gene product matters. So this is what the
00:16:01.15 rare, inherited disease told, that even a 50% gene
00:16:05.00 dosage, one functional gene instead of two functional
00:16:08.22 gene copies, even a 50% gene dosage is a big problem in
00:16:14.24 the long lifespan of humans. Even though one can get
00:16:19.20 often to early adulthood, one cannot keep going
00:16:23.20 throughout life. And the thing that's going wrong is the
00:16:28.08 ability of systems such as the immune system to keep
00:16:32.16 replenishing itself, so the cell proliferation ability is
00:16:36.25 diminished. But that's a rare disease. Is this relevant to
00:16:42.21 what happens in the vast majority of humans who do not
00:16:45.15 have this rare disease, informative as it is? But now that
00:16:50.11 gave us strong clue to look. And so, what's the situation
00:16:55.26 for humans who are not ostensibly carrying any known
00:16:59.22 genetic disease, what's the effect of common variations in
00:17:04.29 how their telomeres are maintained in humans? Variations
00:17:08.16 caused by, for example, nongenetic differences between
00:17:12.25 individuals? So, to put it in perspective, a very interesting
00:17:18.19 observation was made in 2003 by Cawthon and
00:17:22.04 colleagues, and they reported in a cohort of about 140
00:17:26.19 unrelated people aged 60 years and older, when they had
00:17:32.17 shorter telomeres in their white blood cells, which are the
00:17:36.26 cells that are easily analyzed, and a blood sample taken
00:17:40.16 from a healthy individual can be analyzed for its white
00:17:43.07 blood cell telomeres and the average telomere length in
00:17:46.16 those cells... it was found that such individuals with
00:17:49.27 shorter blood cell telomeres have higher mortality rates
00:17:53.10 than those in the same cohort of people with longer
00:17:57.23 telomeres in those white blood cells. And specifically, the
00:18:01.23 shorter telomeres were associated with a three-fold higher
00:18:04.09 mortality rate from heart disease, and an eight-fold higher
00:18:07.26 mortality rate from infectious disease, and that's
00:18:11.05 interesting, given what I just told you about the fact that a
00:18:13.29 50% gene dosage, compared with the normal 100% gene
00:18:19.01 dosage of telomerase RNA, in people with the rare
00:18:22.29 disease, dyskeratosis congenita, that that was enough to
00:18:29.02 make their bone marrow depleted, and so they couldn't
00:18:32.00 fight off infections, in fact they die of infections. So that's
00:18:36.11 a very interesting observation in light of the genetic
00:18:41.00 observations. And indeed, for all causes of mortality in this
00:18:47.03 elderly cohort of people who were followed for 17 years
00:18:51.09 subsequent to the time their telomeres were analyzed,
00:18:56.06 overall, from all causes, the people with the shorter
00:19:00.07 telomeres had higher mortality rates, in other words,
00:19:04.09 poorer survival, compared with those with the longer
00:19:07.09 telomeres. So the telomere length was measured from
00:19:12.07 samples that had been stored away in the freezer, and
00:19:17.16 then 17 years later, it was taken out and analyzed, and
00:19:21.09 these comparisons were made, that it was the
00:19:23.22 subsequent 17 years that were related to whether the
00:19:28.20 telomeres at the beginning of the 17-year period had been
00:19:31.25 shorter or longer. So this was, if you will, a prospective
00:19:37.19 kind of study. They shorter telomeres predicted higher
00:19:42.17 mortality rates. But what caused what? Were the
00:19:48.00 telomeres shorter then because they were already fighting
00:19:50.15 off some of these diseases, and perhaps there'd been
00:19:52.24 more turning over of cells, and telomeres had become
00:19:56.05 shorter already, because people were already prone to
00:20:00.07 these sorts of mortality causes for example? Or was it the
00:20:03.26 other way around? Were the telomeres shorter, and did
00:20:06.16 that make the people less able in the subsequent 17
00:20:11.21 years to avoid mortality from these causes, and indeed, all
00:20:15.23 causes? These results did not say what came first or what
00:20:21.19 caused what. So now I want to tell you about some more
00:20:25.21 recent experiments which start to give one kind of
00:20:29.14 indication of causality in this otherwise, we know, quite
00:20:34.14 complex question of what is causing susceptibility to the
00:20:40.02 diseases of aging. And this was a collaboration which
00:20:43.23 involved studying chronic life stress, and relating that to
00:20:47.11 cellular aging (which in this case, will be related to
00:20:50.26 telomere maintenance) and risks of cardiovascular
00:20:53.24 disease. And our group collaborated with the group of Dr.
00:20:56.29 Elissa Epel at the University of California, San Francisco's
00:21:00.26 psychiatry department, and a number of other colleagues
00:21:04.26 who are all listed here. So now I want to tell you about
00:21:08.26 those findings, which are now done with human beings in
00:21:14.13 vivo, participants in these clinical studies. So the study
00:21:20.01 design was to study chronic psychological stress, and so
00:21:24.09 62 healthy, premenopausal women, aged 20-50 in this
00:21:28.10 group, who were the biological mothers of either a healthy
00:21:31.27 child (the control mothers) or a chronically ill child (a group
00:21:38.04 of caregiving mothers), they were studied. And so first of
00:21:42.02 all they did a standardized questionnaire which has had a
00:21:45.11 good track record as being an assessment of perceived
00:21:49.07 stress, and then the other parameter measured that I'll first
00:21:54.07 talk about was the number of years that the caregiving
00:21:56.28 mothers had been caregiving mothers, that was analyzed,
00:22:00.08 and a number of others that I will allude to later. So first of
00:22:03.28 all, the study design was to assess the perceived stress
00:22:09.09 for the whole group, both the control and the caregiving
00:22:12.28 mothers, and then the number of years of the situation of
00:22:17.15 being in this chronically stressful situation, being the
00:22:23.15 caregiver of their child, who's chronically ill from a variety
00:22:27.05 of causes. So, three markers of cellular aging were
00:22:32.27 looked at: telomerase activity, telomere length, and also a
00:22:35.27 measure of cellular oxidative stress, which is a ratio of two
00:22:40.10 compounds, F2-isoprostanes and vitamin E, and that was
00:22:43.14 just taken as an assessment of the oxidative stress
00:22:48.13 situation physiologically in these individuals. We'll focus
00:22:52.23 on telomerase activity and telomere length. So, this was
00:22:57.02 the study design, as I just went through. And so the
00:23:03.18 questions were: Were the level of perceived stress across
00:23:06.18 both groups of mothers and the duration of caregiving in
00:23:10.10 the caregiver group... were those quantifiable parameters
00:23:14.27 related to markers of cell aging (and the three were
00:23:18.03 telomerase activity, telomere length, and cellular oxidative
00:23:22.10 stress)? Now, just to show you how we measured
00:23:25.08 telomerase activity, what we did was we measured
00:23:28.04 telomerase activity in the white blood cells of these
00:23:31.06 individuals. These are all healthy individuals, but they
00:23:34.11 gave blood samples. One could analyze telomerase
00:23:37.07 activity, and this is the telomerase activity gel, we used
00:23:40.08 three different concentrations always to make sure that
00:23:43.02 we're in the linear range, comparing it with internal
00:23:45.27 controls of various kinds, and a reference sample, which
00:23:49.11 is shown here, and then we quantified all these bands,
00:23:52.21 made sure we're in the linear range, and related it back to
00:23:55.24 the number of viable cells. So we could simply treat
00:24:00.11 telomerase activity in white blood cells as a quantitative
00:24:05.01 parameter, a continuously varying, quantitative parameter.
00:24:09.05 And so across the group, you find typically the lowest and
00:24:12.18 the highest, it's about 20-fold difference, and across the
00:24:15.25 group, there's a log normal distribution of telomerase
00:24:18.12 activities per cell in the different humans in the studies,
00:24:24.02 and we've looked at that in a number of studies and
00:24:26.05 found that same thing. Just to give you a little bit more
00:24:30.06 detail, the oxidative stress assessment came from looking
00:24:35.00 at the ratio of isoprostanes per milligram of creatinine
00:24:38.05 (that's to normalize), over vitamin E, that sort of net
00:24:42.28 oxidative stress assessment, if you will. And this kind of
00:24:47.05 ratio has been used as one kind of marker of oxidative
00:24:50.05 stress and antioxidant defenses that are current in that
00:24:54.23 individual. So what was interesting was that right away
00:24:58.21 one could see that, quantitatively, the worse the
00:25:02.17 perceived stress (the higher the score of perceived stress,
00:25:05.26 that is, the worse it was), the lower was the telomerase
00:25:09.05 length, the lower was the telomerase activity by this assay
00:25:13.16 I've told you about, and actually the worse, the higher
00:25:17.03 was the oxidative stress index. Right across the range,
00:25:20.14 across the entire sample that included the control mothers
00:25:25.12 and the caregiving mothers. Very interestingly, the number
00:25:29.07 of years of caregiving, and obviously this was now in the
00:25:32.07 caregiving group, similarly was related in just the same
00:25:36.29 way, so the number of years of caregiving was related
00:25:40.25 directly to how much shorter the telomeres got, how much
00:25:43.24 lower the telomerase was, and how much higher the
00:25:46.22 oxidative stress index was. So this, just to show you a
00:25:56.06 couple samples of data, here's the high stress quartile,
00:25:59.21 and this was corrected very carefully against all sorts of
00:26:03.00 other parameters in this very well-controlled group, and
00:26:06.10 these relationships didn't go away. Here for example are
00:26:08.21 the data compared for the high stress and the low stress
00:26:13.28 quartiles for example, and you can see here's the
00:26:17.00 average, and here's the range for the high stress quartile,
00:26:20.26 and there's less telomerase, about half the amount of
00:26:23.22 telomerase on average as there is in the low stress
00:26:26.22 quartile. And again, the error bars say that this was quite a
00:26:30.18 significant observation, taken together with the number of
00:26:34.26 individuals analyzed. So this was a striking relationship, as
00:26:40.11 was the relationship of telomere length. Now, I'm just
00:26:43.09 going to point to the error bars, they're very small here.
00:26:46.05 This is the high stress, this is the low stress quartile, you
00:26:49.24 can see these are very different. Here we've controlled
00:26:52.28 for age and body mass index for example. Controlling for a
00:26:56.13 lot of other factors, one still cannot make this difference in
00:27:00.13 the high stress versus the low stress go away, so we are
00:27:06.14 left with the conclusion that the strong variable here is the
00:27:12.06 stress level. Here's the oxidative stress now. The high
00:27:16.04 stress quartile, the low stress quartile, again, distinct. So,
00:27:21.18 stress perception and caregiving duration are linked to
00:27:25.19 cell aging markers, as assessed by telomerase, telomere
00:27:30.03 length, and oxidative stress. So the idea of cell aging is,
00:27:33.06 of course, the more the cell has gone through replicative
00:27:36.17 divisions, then the more aged it is in the sense of going
00:27:39.13 down towards critically short telomeres, then that would
00:27:43.24 be reflected in lower telomere length and potentially lower
00:27:46.24 telomerase. Oxidative stress is going to assess the
00:27:50.17 damage to the molecules in the cells, another measure of
00:27:54.11 their potential aging. Now what are the causal directions,
00:27:59.04 what are the mechanisms? I'll give you two short kinds of
00:28:03.20 indications. First of all, what about the causal direction?
00:28:07.11 So we're seeing these associations, what caused what?
00:28:11.20 Now the number of years of caregiving was a very
00:28:13.28 important thing to think about. So this was the plot of that.
00:28:19.05 This is the number of years of the caregiving situation,
00:28:24.27 and you can see it ranged from actually one year in some
00:28:28.18 of the mothers, some of the mothers had had four, some
00:28:30.29 six, eights, and some had all the way as long as 12. And
00:28:34.25 that was, as you see, quite strikingly related to the
00:28:38.23 shortness of the telomeres, so the ones who had fewer
00:28:41.14 years had longer telomeres on average than the ones
00:28:44.00 who had more years. A similar relationship held for the
00:28:47.10 telomerase activity levels. So this gave us an important
00:28:53.07 kind of indicator, that chronic stress is what's wearing
00:28:57.02 down the telomeres, because of this number of years of
00:29:00.08 caregiving, that objective, stressful situation, being
00:29:03.19 directly related. So, we have one arrowhead in this
00:29:07.26 otherwise no doubt very complex set of interactions, and
00:29:11.20 that is chronic stress (this is now years of chronic stress of
00:29:16.03 a particular kind characterized by the difficulties of
00:29:20.18 caregiving in an unpredictable and often uncontrollable
00:29:24.14 situation)... chronic stress is causing lower telomerase and
00:29:29.07 shorter telomeres, and one could predict that the lower
00:29:32.13 telomerase is in fact leading to the shorter telomeres. Now
00:29:38.24 we know from our biological studies that such effects in
00:29:43.09 normal cells will reduce the ability, eventually, of cells to
00:29:47.05 replenish themselves. So that's an important piece of the
00:29:52.00 picture, because for many model systems and biological
00:29:55.29 studies of cells, that relationship we know is the case. So
00:30:01.00 in whole humans we're finding this arrowhead is in that
00:30:05.15 direction in this particular setting of the chronic caregiving
00:30:09.17 situation. Now, what about mechanisms? In such
00:30:13.12 individuals, when their stress hormones were measured,
00:30:17.06 so these are stress hormones that are produced in
00:30:20.03 response to the brain dealing with this chronic stress
00:30:24.08 situation, cortisol, epinephrine, norepinephrine levels are
00:30:28.04 higher in highly stressed individuals, they're also higher in
00:30:33.03 the low telomerase half of people than in the high
00:30:36.10 telomerase half of people. In other words, you had worse
00:30:39.06 stress hormones if you had low telomerase than if you had
00:30:43.18 high telomerase. This is not implying causality, but the
00:30:47.05 relationship is there, and so it's going to be very
00:30:50.03 interesting to ask whether these stress hormones indeed
00:30:53.16 cause the lower telomerase that is observed in the white
00:30:57.07 blood cells of these individuals. So now we have a new
00:31:02.15 connection here, which we can think of in this way. We
00:31:05.04 think of a signal input, if you will, to the brain, now that is
00:31:08.19 the chronic stress. Now thereafter, things will get very
00:31:12.07 complex, we'll have all sorts of brain-body interactions, we
00:31:16.06 can think of them as signal integration and processing,
00:31:18.25 but a readout, a very quantitative readout, is telomerase is
00:31:21.13 lower, telomeres are shorter. The question is, does that
00:31:27.03 matter at all? Is there any impact on disease? Fortunately,
00:31:33.16 in this study, a great many other parameters were
00:31:36.29 measured. So let's think about cardiovascular disease.
00:31:41.07 What do we know about that? Well, a great deal is
00:31:44.10 known about the risk factors for cardiovascular disease.
00:31:48.26 For example, in one of the largest epidemiological studies
00:31:52.17 of risk factors that's been undertaken, 29,000 people in
00:31:57.02 52 different countries in six different continents, six major,
00:32:04.08 prominent factors for cardiovascular disease risk were
00:32:08.25 shown to be this list here, in this study published by Yusuf
00:32:11.23 et al. in 2004. And they're all the ones you expect:
00:32:15.19 smoking, poor lipid profile, high blood pressure, diabetes,
00:32:18.17 abdominal obesity, psychological stress. Psychological
00:32:21.21 stress. Ahh, now this was the same kind of psychological
00:32:26.06 stress that's the kind that had been measured in this study
00:32:29.17 I just told you about, the mothers and caregiving mothers.
00:32:32.28 So actually this was the same kind of measure, and I just
00:32:35.15 told you that relationship had been found, that is, lower
00:32:40.20 telomerase activity, higher oxidative index, shorter
00:32:44.06 telomeres, had been found associated with the higher
00:32:48.21 levels of psychological stress, and the longer the duration
00:32:52.24 of the psychological stress. So we had one, but actually
00:32:56.08 in these very well-designed studies, all of these had also
00:33:00.04 been measured, and so in fact, there were measures for
00:33:03.19 either the risk factor itself or, in one case, a surrogate for
00:33:07.02 it. One of the risk factors is diabetes, that's a known risk
00:33:11.16 factor for cardiovascular disease. All the individuals in this
00:33:15.08 study I told you about were all healthy, so by definition,
00:33:18.08 nobody had diabetes, but in fact, fasting insulin and
00:33:21.21 glucose were measured, and those are, if they're high, a
00:33:24.28 risk factor for diabetes. So all of these risk factors, or in
00:33:30.01 this case, a surrogate for the risk factor, were all in place
00:33:34.29 in this study. So the question was, well, since these three
00:33:39.19 had been associated with the level of psychological
00:33:44.10 stress and the duration of psychological stress, what
00:33:47.11 about these? Interestingly, it turned out that the one thing
00:33:52.15 that emerged for all of these was, in this study which has
00:33:57.24 been published, lower telomerase activity in the white
00:34:01.08 blood cells. So that went with smoking, it went with the
00:34:05.01 bad cholesterol/blood lipid profiles, it went with worse
00:34:10.13 cardiovascular activity profiles, with higher fasting
00:34:16.00 glucose, higher adiposity, and as we talked about, higher
00:34:19.06 and longer psychological stress. So the one parameter
00:34:23.10 that emerged in this particular study was lower telomerase
00:34:27.14 activity, now which, as we have talked about, is
00:34:31.22 predicted to lead to lower telomere length, but that didn't
00:34:35.21 emerge in this particular study, perhaps because the
00:34:38.29 individuals were relatively young. So, just to show you for
00:34:45.16 smoking, here is the range of telomerase activity per cell
00:34:51.27 in the white blood cells for the nonsmokers, you see it
00:34:54.07 follows a broad distribution, in fact, this is a log normal
00:34:57.07 distribution for the whole group. Here are the smokers.
00:35:01.09 They're all very, very low telomerase, in fact, the p-value,
00:35:04.22 the probability that this would be observed just by
00:35:07.18 chance, is very, very low, so telomerase was very low in
00:35:11.20 the smokers. There's no causality implied here, this is just
00:35:15.02 the association. Here's a very interesting observation for
00:35:20.13 heart function. So one can take all the telomerase activity
00:35:25.26 numbers for the entire group and simply divide them down
00:35:29.02 the middle, it's called a median split, into those who have
00:35:32.25 low telomerase and those who have high telomerase.
00:35:37.01 Now, some laboratory analyses of these individual
00:35:41.05 participants were done. People sat for half an hour or
00:35:47.15 longer, and then their resting heart rate was measured,
00:35:50.27 and so here's the value for the high telomerase half, and
00:35:53.25 here's the value for the low telomerase half. You can see
00:35:56.06 right away that the low telomerase half have higher
00:36:00.02 heartbeat rate, just even resting, then the lower
00:36:03.15 telomerase half. The pulse pressure, the blood pressure,
00:36:08.03 is higher for the low telomerase people than the high
00:36:12.21 telomerase people, so in other words, these are less
00:36:16.10 healthy. There's a parameter called "high frequency heart
00:36:19.23 rate variability," basically the variability is how well the
00:36:23.17 heart is adapting to changing needs, so when it's high,
00:36:26.29 that's good, it's adapting well. So you can see it's higher
00:36:30.12 in the people with high telomerase than it is with the low
00:36:32.15 telomerase group. Everybody's sitting resting. Then, an
00:36:37.15 interesting other thing was done in these individuals. They
00:36:44.07 were asked to undergo a series of laboratory
00:36:49.04 psychological stressors, in a very controlled setting. So
00:36:52.24 they are asked to anticipate giving a short videotaped
00:36:56.21 speech, to give the speech, and then to do some very
00:36:59.20 unpleasant mathematics in a highly controlled
00:37:02.26 environment in which individuals who are trained
00:37:06.07 personnel look at them stony-faced, and so, that normally
00:37:11.09 elicits a very uncomfortable response. Interestingly, how
00:37:15.29 people responded depended on whether they fell into the
00:37:20.04 high or low telomerase group. So I'll just show you the
00:37:23.10 curves, it's quite striking. So what you expect is, of
00:37:26.12 course, your heart rate will go up as you become more
00:37:30.23 nervous and uncomfortable with this deliberately
00:37:34.01 uncomfortable, rather unpleasant task. And you can see
00:37:36.29 that the low telomerase people, their heart rate went way
00:37:39.10 up, much more (it was already resting higher), went up
00:37:41.29 much more in response than the high telomerase people.
00:37:46.03 Similarly, their pulse pressure went much higher, and then
00:37:51.14 eventually came down somewhat, than the high
00:37:55.21 telomerase people. And the heart rate variability showed a
00:37:58.07 nice, healthy response in the high telomerase individuals,
00:38:03.06 but a much less healthy lack of ability to adapt in the low
00:38:08.10 telomerase people. So, simply splitting all of the
00:38:12.21 telomerase activity measurements into just two halves, the
00:38:16.21 high half and the low half, showed very different heart
00:38:20.16 functions. And in all cases, the difference was that the
00:38:25.10 low telomerase group always had the unhealthy response
00:38:28.24 that is predicted to lead to have more of a cardiovascular
00:38:32.17 disease risk. So what we've seen then, in summary, the
00:38:37.11 low telomerase alone, even in the absence of obvious
00:38:41.01 telomere shortening, which may well happen later but
00:38:43.16 hasn't happened in this group, is associated with six major
00:38:47.06 risk factors, including chronic psychological stress for
00:38:51.03 cardiovascular disease in people, and so that raises an
00:38:55.18 interesting question of, is telomerase status in normal cells
00:39:01.02 of people an indicator of their disease risk for, for
00:39:05.08 example, cardiovascular disease? Now indeed, telomere
00:39:09.23 shortening has been seen a lot as something that is being
00:39:15.11 associated with disease risks, and indeed disease is
00:39:18.23 incident now in more and more cohorts. So just to finish
00:39:24.27 the idea, the idea is that if you have lower telomerase, we
00:39:28.03 know that will be one of the things that will help drive
00:39:30.27 telomere length down, because telomere length
00:39:33.03 maintenance will be less able to be at the optimum level if
00:39:38.10 telomerase is down. So that, as we know, reduces the
00:39:43.20 ability of cells to replenish themselves. Now, that then
00:39:53.14 makes us step back and say, well, what about telomerase
00:39:58.12 and genetic components? I've just talked to you about a
00:40:02.03 clearly nongenetic component, years of stress induced by
00:40:07.18 a chronic caregiving situation. Here, let's consider known
00:40:13.05 genetic defects. Mouse telomerase has been knocked
00:40:17.03 out in model organisms, and I told you about the effects of
00:40:21.10 losing function of half the telomerase gene dosage in
00:40:25.23 humans. Again, telomerase activity goes down and
00:40:29.10 telomere length maintenance is going down, and in this
00:40:32.00 case, the cause is known to be less telomerase, and
00:40:36.18 telomere length does indeed go down. And indeed, one
00:40:39.16 does see reduced ability of cells to replenish themselves
00:40:43.16 in these situations where, genetically, telomerase has
00:40:47.29 been knocked down, so one knows for sure that that is
00:40:51.00 leading to these effects. Now, what about disease
00:40:56.21 impact? Indeed, the genetic defects do lead to diseases,
00:41:03.07 and in mouse models in which telomerase is knocked out,
00:41:06.26 there is indeed evidence for diseases of the kind that can
00:41:11.29 include propensity for cardiovascular disease. Now,
00:41:16.27 what's been seen clinically for many years is chronic
00:41:19.24 stress, which we can think of as that signal input, clearly
00:41:22.27 has disease impact. This has been seen in a great many
00:41:28.04 different studies. Now we've added a new question: Does
00:41:34.13 that occur via lower telomerase? And that is the open
00:41:38.07 question. So as I said, what we know is we have one
00:41:42.10 arrowhead here, we know chronic stress indeed can
00:41:45.19 cause less telomerase and worse telomere maintenance,
00:41:50.12 shorter telomeres, which we know from our biological
00:41:53.11 studies does in fact affect whether cells can replenish
00:41:59.20 themselves. We know from the genetic studies that that
00:42:03.13 can lead to disease impact, but the question is, is this the
00:42:11.14 case in the human studies I've described to you? Is this
00:42:17.26 really what's causing the disease impact, via the lower
00:42:22.12 telomerase? Or is it that the chronic stress actually has its
00:42:27.14 disease impact because of unrelated reasons? Or, more
00:42:31.09 likely, is it a combination of the two? So that is where
00:42:34.26 things are standing right now. Finally, just let me show you
00:42:39.26 a lot of data that have been accumulating in the last few
00:42:43.25 years, in which low telomere length is being more and
00:42:48.04 more linked to rather common diseases of aging, such as
00:42:52.20 cancer, cardiovascular disease, vascular dementia,
00:42:56.19 various degenerative conditions, diabetes, and in fact,
00:43:00.02 risk factors overall for chronic disease. And a great many
00:43:03.19 studies have linked shorter telomeres in white blood cells
00:43:08.21 to these rather common diseases of aging or risk factors
00:43:13.02 for such diseases. So in a great many cohorts, low
00:43:17.27 telomere length is certainly linked to these diseases. The
00:43:21.20 causality of course is not determined by any of these
00:43:24.28 studies, but we think that we may have a clue from our
00:43:30.06 finding that chronic stress will have effects on lowering
00:43:33.19 telomerase, which is expected to lead to lower telomere
00:43:37.00 length. So back to this general statement about genetic
00:43:42.23 and nongenetic aspects of aging in humans and disease
00:43:47.00 susceptibilities that lead to mortality in humans, the
00:43:51.05 general observation is that elderly subjects demonstrating
00:43:54.26 exceptional longevity have generally been spared these
00:43:59.05 major age-related diseases, such as cardiovascular
00:44:02.16 disease, diabetes, and cancer, which are responsible for
00:44:05.19 most deaths, and for which I've just shown you the kind
00:44:09.11 of evidence that is beginning to accumulate suggesting
00:44:13.28 that there are nongenetic (as well as, of course, some
00:44:17.06 genetic) components for these diseases. And so these
00:44:26.21 nongenetic as well as genetic causes are obviously very
00:44:31.12 interesting, since these major diseases are responsible for
00:44:35.06 most of the deaths in the elderly. So how do we age? The
00:44:41.16 question has come from what we've learned in the lab
00:44:45.00 about cells and molecules: telomerase, telomeres, how
00:44:49.29 their continued maintenance affects abilities of cells to
00:44:53.19 maintain themselves. In animal models, one sees that, if
00:44:57.09 it's compromised, it can lead to cell death and mortality of
00:45:02.05 the organism. That knowledge has been carried to the
00:45:06.00 understanding of some of the diseases of aging and
00:45:11.11 back, and so this continuous sort of cycle of knowledge
00:45:16.25 and understanding, I think is going to an important part of
00:45:20.15 how we address the question of how do we age. So, let
00:45:26.02 me summarize with a little metaphorical picture. The
00:45:29.01 summary is that it's emerging that shortening of telomeres
00:45:31.28 in human cells is associated with shortening of life. So if
00:45:36.06 we think of a chromosome and the telomeric structure at
00:45:40.02 its end, I like to think of this structure as being a beautiful,
00:45:43.04 elaborate tree, and what we're trying to understand are
00:45:48.10 the sorts of things that lead this beautiful tree to erode
00:45:51.27 down to a stump. Thank you.
00:45:57.22

Videos in this Talk
  • Part 1: The Roles of Telomeres and Telomerase
    Part 1: The Roles of Telomeres and Telomerase
    Audience:
    • Educators of H. School / Intro Undergrad
    • Educators of Adv. Undergrad / Grad
    • Student
    • Researcher
  • Part 2: Telomeres and Telomerase in Human Stem Cells and in Cancer
    Part 2: Telomeres and Telomerase in Human Stem Cells and in Cancer
    Audience:
    • Educators of H. School / Intro Undergrad
    • Educators of Adv. Undergrad / Grad
    • Student
    • Researcher
  • Part 3: Stress, Telomeres and Telomerase in Humans
    Part 3: Stress, Telomeres and Telomerase in Humans
    Audience:
    • Educators of H. School / Intro Undergrad
    • Educators of Adv. Undergrad / Grad
    • Student
    • Researcher
Speaker: Elizabeth Blackburn
Total Duration: 2:01:21
Recorded: June 2008
All Talks in Genetics and Gene Regulation
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Talk Overview

Telomerase, a specialized ribonucleprotein reverse transcriptase, is important for long-term eukaryotic cell proliferation and genomic stability, because it replenishes the DNA at telomeres. Thus, depending on cell type telomerase partially or completely (depending on cell type) counteracts the progressive shortening of telomeres that otherwise occurs. Telomerase is highly active in many human malignancies, and a potential target for anti-cancer approaches. Furthermore, recent collaborative studies have shown the relationship between accelerated telomere shortening and life stress and that low telomerase levels are associated with six prominent risk factors for cardiovascular disease.

Speaker Bio

Elizabeth Blackburn

Elizabeth Blackburn

Dr. Blackburn is a leader in the area of telomere and telomerase research. She discovered the molecular nature of telomeres-the ends of eukaryotic chromosomes that serve as protective caps essential for preserving the genetic information – and discovered the enzyme telomerase, which replenishes telomeres. Blackburn is currently a Professor in the Department of Biochemistry and… Continue Reading

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Related Resources

Blackburn, E.H., Greider, C.W., and Szostak, J.W. Telomeres and telomerase: the path from maize, Tetrahymena and yeast to human cancer and aging. Nature Medicine 12:1133-1138 (2006).

Epel, E.S., J. Lin, F.H. Wilhelm, O.M. Wolkowitz, R. Cawthon, N. Adler, C. Dolbier, W.B. Mendes, and E.H. Blackburn. Cell aging in relation to stress arousal and cardiovascular disease risk factors. Psychoneuroendocrinology 31: 277-287 (2006) [Epub: Nov. 17, 2005].

Epel, E.S., E.H. Blackburn, J. Lin, F. Dhabar, N. Adler, J. Morrow, and R. Cawthon. Accelerated telomere shortening in response to life stress. Proc Natl Acad Sci USA. 101: 17312-15 (2004).

Blackburn, E.H. Switching and signaling at the telomere. Cell. 106: 661-673 (2001). Blackburn, E.H. Telomere states and cell fates. Nature 408 (6808): 53-56 (2000).

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