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Welcome to the second part of this three-part lecture
series that I'll be giving on telomeres and telomerase. In
part two, I'm going to discuss telomeres and telomerase in
human cells, and particularly I'm going to emphasize the
setting of cancer cells. Now you may recall from the first
lecture that the function of telomerase is to maintain the
telomeres and prevent them from shortening as cells
divide, because telomere shortening would otherwise
occur in the absence of telomerase, to compensate for
the shortening processes. And so, maintaining telomeres
allows the cells to keep on dividing. In human cells, we
can also see though that telomerase itself is protecting
telomeres, and so I want to show you one kind of
experiment for that. And the conclusion is going to be that
it's not just the bulk telomere length that matters, but the
presence of telomerase can determine whether a
telomere is seen by the cell as sufficiently long or not. The
experiment I'll show you was done in cultured human
cells. These were not cancer cells, and these were cells
that have normally extremely, extremely low levels of
telomerase, effectively, for our purposes, essentially no
measurable telomerase activity, and certainly their
telomeres are not maintained. Now I've shown you in very
simple diagrammatic form human telomerase, so this
would be the template sequence of the human
telomerase RNA, and this is the overhanging G-rich
strand. It's actually usually longer than this, but I've just
shown it as a simple diagrammatic form here. And this of
course is the duplex telomeric DNA that will be consisting
of hundreds or thousands of telomeric repeats, as you go
in toward the chromosome interior. And just as I showed
you for Tetrahymena telomerase, the template region is
copied, and so the DNA... for example, a DNA with three
Gs, two Ts, and an A, would sit down here on the
template, and then nucleotides would added, extending
along the template, thereby lengthening the DNA in this
reverse transcriptase reaction that telomerase carries out.
So what was done was to compare cells in which
telomerase was being expressed or not being expressed,
and look at the growth of the cells and the telomere
length. Now the protein TERT is the core protein that has
this enzymatic activity: the reverse transcriptase activity,
and probably other activities, as I will allude to. And so, in
these experiments, a particular mutation was made on the
TERT protein, it happened to be a very small change of a
few amino acids added to the very C-terminus, and what
this does is it doesn't affect the enzymatic activity, but it
does affect the ability of this enzyme to elongate
telomeres in cells, and it's called a hypomorph because
that refers to the fact that it has an insufficient function,
but it showed something useful. Now here's the
experiment, and I'm going to walk you through this rather
complex-looking slide. First of all, I want to show you a
growth curve of human fibroblasts in culture. What
normally happens is, if we look at the... this is cumulative
dilutions, which is just an operational term telling you how
many cell divisions are going on, and this is the number of
days. So if you culture human fibroblasts, normally what
you find is that the cells will continue to multiply for a
while, and then they'll cease multiplying any further, so the
curve flattens out, so you see they've undergone
something like 50 or so divisions. And if you put in a
control vector, which would be important, you get the
same curve. But if you put in the test vector, which
actually is expressing this form of the human telomerase
core protein TERT, because the fibroblast cells naturally
have enough telomerase RNA and the other components
of telomerase, all they're missing is the TERT, you just
have to add this in, and now you can restore telomerase
activity. But what's very interesting is the telomeres and
the cell growth. What you can see is, first of all, the cell
growth has been greatly extended. We've now made
these cells very much elongated in their lifespan
compared with the controls or the parental cells. Now let's
look at the telomeres. If we look at the telomeres in these
cells, the controls, we find that they're gradually becoming
somewhat shorter, and at this point here, they've pretty
much ceased to divide, so we're out here, they pretty
much cease dividing. So the telomeres on average are
about this long, and the cells have picked up the signal,
they've said the telomere length is shorter here than here,
and they've picked up the signal, and they've said we're
not going to divide any further. Now we've added the
hTERT. Now actually what happened was that, I told you
the cells continue to grow a long, long time. The
telomeres shorten, and then they steady out at some
much shorter length, so through here and through here,
they're actually growing quite well, but they're going with
much shorter telomeres. So in other words, these cells
can keep dividing for a long time with very short
telomeres, and the difference was they had telomerase
being expressed. So we used a trick just to separate out
the telomere lengthening property of telomerase from its
ability to stabilize telomeres, and we saw that in fact if you
have this telomerase present throughout, even though
the telomeres maintain short, they are perfectly stable,
and the cells can keep dividing. So this is the second
piece of evidence, I showed you the first piece of
evidence for you in yeast systems in the first part of this
lecture series, and now this is the second piece of
evidence that telomerase is having a protective function,
in this case, it's in human cells, and it's stabilizing
telomeres that otherwise would've been too short in its
absence. This is not unique to human cells, it's been seen
in yeast systems as well experimentally. So I just talked to
you about normal cells, and I told you that, in those
human fibroblasts grown in culture, there's very little
telomerase. So, now let's talk about where do we normally
find telomerase in human cells? Well, if you look in cells,
you find that it actually is on at times when the cells are
greatly proliferating during fetal development, and it does
remain active in certain proliferative cells. It's also found
active in stem cells, in cells that are activated to
proliferate, such as lymphocytes proliferating under the
response to, for example, a pathogen. One finds stem
cells, for example, in hair follicles; those have telomerase.
So one does find telomerase in cells that are stem cells
and various sorts of cells that are induced to proliferate.
And in fact in most other cells, one can find telomerase,
but it's in very low levels. So initially people thought there
was no telomerase in normal epithelial cells or fibroblasts
or endothelial cells, but closer scrutiny showed that in fact
there were real, definitely low and very downregulated,
but real levels of telomerase. And in the third part of this
three-lecture series, I will talk more about the telomerase
in the normal cells of people and tell you about some in
vivo studies that have been done. Now, cancer cells.
Cancer cells are infamous for their ability to keep on
multiplying. Now, they can do this for a variety of different
reasons. They lose their system of checks and balances
that prevent them from overmultiplying, and that's
because of a lot of genetic and epigenetic changes that
have taken place in their progression to become a
malignant cancer cell. Now, they are immortal cells, and
indeed, as you might expect, telomerase is on in these
cells, and in fact it's very high in the vast majority of
human cancers, particularly as they've got to the invasive
stages. And that makes a lot of sense based on what I
just told you, because if the cells are to keep on
multiplying, then they have to keep on replenishing their
telomeres. Now I'll you some more recent results in the
last few years, which also suggest telomerase may be
doing other things in human cancer cells. It is certainly
maintaining the telomeres, and that is important, but there
may be other functions as well. So I'll tell you some newer
findings about that. So, let's just recapitulate what I said
about where we find telomerase in humans. So it keeps
telomeres elongating and replenished, and therefore cells
can keep dividing in stem cells and, of course, I didn't
mention germ cells. Now of course we wouldn't be here if
we didn't have maintenance of telomeres from generation
to generation. Telomerase indeed is active in germ cells,
as well as stem cells of various kinds. As I said, it's
detectable in many normal cell types, and highly active in
the great majority of human cancers. And by the way, in
some of those human cancers in which you don't find the
high telomerase, one actually often finds this ALT
mechanism, but it's only a particular subset of cancers in
which one finds ALT being a prominent means of
maintaining telomeres. The great majority of the common
human tumors have highly active telomerase. So,
telomerase is highly active in human tumors. So one could
imagine inhibiting telomerase, and this might be a good
target for trying to inhibit the growth of cancer cells, if you
could inhibit telomerase in cancer cells. And so in
investigating this, some interesting things emerged. So
let's just think about what is expected to happen if you
don't have telomerase in the cancer cells. Well, as you
know, cells multiply, and their telomeres will progressively
become shorter and shorter if there isn't telomerase to
counteract that shortening, and so eventually when the
telomeres get too short, the cells would eventually cease
to divide, and the cells respond to those short telomeres
by either what's call the "senescence response," in which
the cells simply won't replicate their DNA anymore, or a
cell death response that can include an apoptotic
response, which involves an active cell suicide program
that can be induced by dysfunctional telomeres. Whether
it's senescence or cell death does depend upon the cell
type. So, the simple prediction, diagrammatically, would
be, if you didn't have telomerase, the cells now would
have shorter and shorter telomeres, and after some
number of divisions, eventually there would be cell death.
Without telomerase, it's been observed experimentally,
typically human cells lose their telomeric DNA at this kind
of rate. I've put 150-200 base pairs per cell division; that's
seen in a number of cultured cells and also in some
cancer cells in which telomerase has been inhibited.
Now, I told you that human telomeres are typically made
of hundreds, even thousands, of copies of telomeric
repeats, they're thousands of base pairs long, so at this
rate, it's going to take quite a lot of cell divisions before
the telomere gets short enough that the cells will have this
response. So that in fact is the prediction, and if you
inhibit the telomerase enzyme using, for example, a small
molecule inhibitor that inhibits the catalytic function of
telomerase that prevents it from carrying out that DNA
polymerase reaction by the reverse transcriptase
mechanism, if you inhibit telomerase in such a way,
indeed this is the observation. There's a gradual
shortening of telomeres, and eventually the cells cease to
divide. So this is seen in cancer cells in culture treated
with such an inhibitor. Now I've put a noted point up here:
but you're still keeping the telomerase ribonucleoprotein
(RNP) level high. You're not depleting the cells of the
enzyme, you are simply rendering that enzyme inactive.
And I make that distinction because of the next results I
want to tell you about. So, what was observed was quite
surprising. If one depleted telomerase, and the particular
way to knock the telomerase RNA down, then one found
there was a very rapid effect on human cancer cells. One
didn't see the long delay ensuing before the effect was
observed. So, now I'll tell you about those experiments.
So, reminding you again, here's human telomerase, it's
copying its RNA template, and of course the enzyme has
the TERT protein and the telomerase RNA, and it's the
RNA that is going to be depleted in these experiments.
How's the RNA depleted? A now commonly used
technique, which is called a knockdown using RNA
interference. Now the particular technique was to express
from a lentiviral vector, which you can introduce efficiently
into cells, a particular sequence which forms a double-
stranded RNA, and I've just shown the corresponding
DNA sequence here, and that double-stranded RNA can
interact with a cellular RNA that matches its sequence,
and eventually cause the breakdown of that RNA
through a complicated process known as RNA
interference. And so, siRNA refers to "short interfering
RNA," because such a short interfering RNA, which
involves introducing two strands of RNA complementary
to the target RNA, that is what causes the breakdown to
occur. Now when we did this experiment, so here's a
control where we're looking at a reference amount, here's
the telomerase RNA, one found that in fact the method of
introducing such a short interfering RNA by this kind of
construct here (the details don't matter), one could in fact
quite efficiently knock down the telomerase RNA in
human cancer cells grown in culture, for example, in
these breast cancer cells grown in culture. And so one
could knock it down almost down to about 10-12% of the
original level that one sees in the controls. So what
happens then? Now, if what I had told you was the case,
that we knock down the telomerase, then we would
expect to have to wait for a long time for the telomeres to
get short enough, if that were the only thing going on,
then we would have to wait before we saw any effect.
But right away, there was an effect on the growth of the
cells. Now just to put this into perspective here, I'll just
walk you through a couple of graphs. Here's the controls
of two kinds, and this is the number of cells here. And so
what you can see is that the cell number goes up and up
and up, as you might expect. The cells are dividing once
every day or two, and so you can see, after about four or
five days after the introduction of the construct that
knocked the telomerase RNA level down, that the cells
that received this are quite quickly growing more slowly
than the controls. So this must be occurring within a
couple of cell divisions. And here are some more controls
in which, here's the empty vector, here is the short
interfering RNA targeting telomerase RNA, and here's a
control version of that, in which it now no longer can
target the telomerase RNA, but everything else is the
same, and as you can see it behaves like the controls.
And so a great many control experiments were done to
show that this was a specific effect specific to knocking
down the telomerase RNA. This was done on bulk,
unselected cell populations. The cells were a melanoma
cell line that normally has very long telomeres. The bulk,
unselected populations is significant because what it
meant was that one could put the siRNA, the agent that
knocks the telomerase RNA down, into the cells at day
zero, and without even any selection, one could get
something like 80-90% of the cells that received this
construct, and in fact the ones that did grow out were
that low percentage that didn't receive the construct. So
in fact the effect is even stronger than what these curves
would indicate, because these ones that are growing out
are largely the ones that just didn't receive the construct.
Now, one of the things that is very important in human
cells to respond to DNA damage such as certain kinds of
telomeric DNA damage is the gene p53. Interestingly,
these effects did not require p53. Here is a pair of cell
lines that are otherwise isogenic. This is a colon cancer
cell line called HCT116, in these cells the p53 is wild-type,
here's the response to knocking down telomerase RNA.
In this otherwise isogenic cell line lacking p53 but
otherwise the same, this response is quantitatively the
same. So this is a different response from a classic DNA
damage response, and in fact we did not see DNA
damage response genes being induced here. We looked
at the telomeres by some molecular probing mechanisms
that allow one to see if the telomeres are uncapped, and
in fact they were not uncapped either. So we see, when
we knock telomerase RNA down, we rapidly see an
inhibition of cancer cell growth, these are cells that
normally have very high telomerase. p53 is not required
for this, suggesting it's not a classic DNA damage
response. And the telomeres, as far as we can see, are
not uncapped, and there's not DNA damage response.
Indeed, the telomeres hadn't had time to shorten
perceptively at all during this short timeframe. So the rapid
knockdown doesn't uncap the telomeres. That's not the
cause of the growth inhibition of these human cancer
cells. But interesting things happen very quickly to these
cells when you knock down telomerase RNA. I'm going to
show you this one experiment in which, in this experiment,
melanoma cells were grown in culture, and the RNA level
was knocked down by a completely different mechanism,
it's called a "ribozyme," and the purpose of it is to cleave
the telomerase RNA, causing it to break down, and so it
has exactly the same effect that I showed you for the
RNA interference: you knock down the telomerase RNA
level. A very interesting thing was seen. These are cells
that are growing in culture, and these are three flasks of
cells that either are control cells that just got the empty
vector. The cells are all growing on the bottom of the
flask, you can't see it. We're just looking at the medium,
the broth, the liquid medium in which the cells are
growing, and it's a nice, pinkish color here in the controls.
But here, in two separate versions of the cell lines that
received the construct that knocked the telomerase RNA
levels down, you can see that the medium has turned a
dark color. These were melanoma cells, so it was of
course very easy to wonder if indeed these cells were
now producing the pigment melanin in high amounts in
this and this but not in the control cells. And indeed,
analyses showed that that was exactly what was
happening. And in fact, a lot of interesting things
happened. These cells became more like their normal
counterpart cells. It was as though they were more
differentiated; they looked more dendritic in form, as
though they had become less cancerous. And indeed
their gene expression profiles had changed. So, knocking
down telomerase RNA also made these cells less
invasive. The more differentiated the cancers are, very
typically, the less invasive they are, and in fact, in
preclinical mouse model systems used, in fact metastasis
was knocked down. And that was seen whether one
knocked telomerase RNA down with the ribozyme, or with
the RNA interference mechanism, but in melanoma
models for cancer, using the experimental mouse system
in the laboratory, it was found that the metastasis is
decreased when the telomerase RNA is depleted. So
knocking telomerase RNA levels down changed the
nature of the cells, and it changed the nature of the cells
well before the telomeres became uncapped. So, as I
said, when you have plenty of telomerase, you expect
cells to be able to multiple indefinitely, and you don't
expect any effects to occur, of removing telomerase, until
a long time has elapsed. So, what we see by this abrupt
knockdown of telomerase level, by knocking down the
telomerase RNA, is we see rapid inhibition of cell growth,
p53 was not required, there was no DNA damage
response or telomere uncapping, and in fact metastasis is
reduced. Metastasis is reduced, what is going on? I briefly
mentioned, we looked at the gene expression profiles in
these cells, and we found that in fact that rapid
knockdown of telomerase RNA and that rapid slowing of
the cell growth is accompanied by and presumably
caused by cell cycle and tumor progression genes being
downregulated. Glucose metabolism is downregulated;
cancer cells typically have high rates of glucose
metabolism. That becomes downregulated in cells that
have less telomerase RNA. And the cells appear more
differentiated, making one wonder if there's a cell
differentiation program that's induced in these cells.
Unexpected changes, many open questions remain, but
these kinds of experiments have led to these
observations here that have two kinds of implications:
One is the scientific implication that telomerase has other
functions that are not solely mediated through its adding
telomeric DNA to the ends of the chromosomes, and the
other implication is that this makes telomerase an
interesting target for potential anticancer therapies. And
the take-home message that I think is most impressive
and unexpected was, there seems to be good reason to
think that high telomerase levels are promoting an
undifferentiated, "stem cell-like" phenotype, a very
unexpected observation. And in fact, now in other
systems, I won't have time in this lecture to go through it,
but there's now evidence that this is really the case, in
even noncancerous cells in certain model organisms
where this has been studied.