So today's lecture talks about the cell cycle, the control of the cell
cycle, and also cell death. So obviously, cell division is
extremely important in multi-cellular organisms.
We've talked a fair bit about control of the cell cycle,
in terms of mitosis and meiosis, in earlier lectures.
Obviously, cell division is necessary in a variety of
circumstances, probably the most obvious is
development. You go from one cell to ten to the thirteenth to ten to
the fourteenth cells. That's accomplished by a tremendous
amount of cell division that goes on over your gestational period.
I've also pointed out, in fact we talked about it last time,
that cell, that wound healing is, in part, a process of cell division.
Fibroblasts for example,
and other epithelial cells, get recruited to divide, in order to
repair damage to a tissue.
And even without damage, there's a lot of cell turnover,
for many tissues. You may not realize this, but your blood,
for example, turns over about every month, so you have to replace all
your red blood cells, and all your white blood cells,
and so on, periodically. Cells in other tissues,
in the skin for example, your skin cells are born,
they migrate up to the superficial layers of your skin,
and then they get sloughed off, and of course, they have to be
replaced. There's a lot of cell division that's going on naturally,
in the process of what we call homeostasis, and to give you an
example of that, if you consider your intestines in a
human, your intestines undergo ten to the eleventh cell divisions per
day. That's a remarkable number. Ten to the eleventh cells dividing
in your intestines per day. If you imagine that a cell in your
intestine is about ten microns in diameter, if you lined up all the
cells next to each other that were born in a given period of time,
in 38 days you would have enough cells lined up end-to-end to go
around the earth, and in a year you'd have enough
cells to make it from the earth to the moon. So you produce a
tremendous number of cells just naturally, in the process of organ
function and homeostasis. Of course we can have too much cell
division, and this is pathological condition, which we typically
associate with cancer. And indeed, many of the factors
that I'm going to talk to you about today, that relate to the normal
control of cell division, and also cell death, are perturbed
in the development of cancer. Cancer, as you almost certainly
know, is a disease of too many cells, and a major factor in that is
excessive cell division. [UNINTELLIGIBLE PHRASE].
So this happens to be a human cancer cell line growing in tissue
culture, and one of the hallmark features of cancer cells is that
they have, in a sense, unlimited and unregulated cell
division. So if you were to watch this movie, which actually comes
from your book, supplementary materials from your
book, you could watch these cells dividing, and they would do so in an
unnatural fashion. Without response to proper growth
factors that would stimulate cell division, they would divide on top
of one another, which normal cells don't do,
and other places and times when other cells are kept in check,
cancer cells are not. So the basic process, which again is well
familiar to you, is to take a single cell and turn it
into two.
And we discussed the very last stages of this in earlier lectures
on mitosis, how the chromosomes get divided from the mother to the two
daughter cells. We also have briefly alluded to the
fact that there's a second, critical event, and you talked about
the mechanisms of DNA replication. The DNA needs to get duplicated so
that it can be properly divided, and this, as you know, occurs during
a particular phase of the cell cycle, known as S-phase, for
synthesis phase. And then there's chromosome
segregation, and this occurs in a distinct phase of the cell cycle
called M-phase, or mitosis phase.
And the development of cells over the course of,
say development, or in wound healing,
can be thought of as a successive iteration of DNA duplication,
DNA replication, and chromosome segregation. So in a sense,
you go from mitosis to S-phase to mitosis to S-phase.
These segments are separated by periods of time when cells are
accumulating the biomaterials that they need, basically to function,
and also to carry out the next phases of the cell cycle,
and these are refereed to as gap phases, G1 for the one that
separates mitosis from S-phase, and G2, the one that separates the
S-phase from mitosis. Now, this is a cyclic process,
one cell gives rise to two, that then undergoes this process again,
and so we think of these things as cycles from M-to-S,
connected by these gap phases, G1 and G2. Now there's another
phase that we haven't told you about, which many of your cells are
actually in right now, and that's a phase called G0.
This is a resting phase, and this can be either permanent or
temporary. Many of your cells, when they're born, will stop
dividing forever. Cells in your brain,
for example, or most cells in your brain, cells of your cardiac muscle,
and there are many other examples. Once they get born,
they undergo mitosis, they go into this resting phase,
and they'll never come back out again.
And that's why it's difficult, for example, to deal with brain
injuries, or spinal cord injuries, because those cells cannot be
recruited back into the cell cycle, they can't make more of them. But
there are other cells in which the resting phase is temporary,
and these cells can then reenter the cell cycle, going back from G0 to G1
and through the process again. And a lot of the progenitor cells
that I referred to up there, in terms of the skin and the blood,
and the intestines, are in that situation.
They're resting, and then they be recruited back into
the cell cycle, in order to make more derivative
cells. It's also useful to consider what the chromosomes are doing in
these different phases of the cell cycle. So in a G1 cell,
if we think about a single chromosome, and we're representing
it by a single-line, although this is of course,
a double-helix. In the G1 phase there's a single,
double-helical chromosome, in S-phase that gets duplicated in
the process of DNA replication, initiation shown here, and it would
be completed so that the single-chromosome would be
ultimately duplicated into two.
And then in M-phase, those two chromosomes get separated
from one another, so these are now joined,
separated from one another, eventually into two daughter cells,
which can then go through this process again.
OK? And much of what we think about, when we think about how the
cell cycle is controlled is to deal with the initiation of DNA
replication, and then the initiation of mitosis.
OK, so this is a cyclical process, a highly ordered process, this is a
representation of the cell cycle, as shown in your book, just so you
know it's in there. Exactly as I described to you,
mitosis and S-phase, where the action is, these gap phases,
G1 and G2, and this little arrow represents G0,
and I've also used this term before, interphase, that represents all the
phases where the DNA is not evident under the light microscope.
It's only evident in mitosis because of DNA condensation,
so the rest of the cycle, G1, S, and G2, are called interphase.
So this is a highly ordered process,
each step preceded by a particular other step, leading to a particular
outcome, events leading to a particular outcome,
and in a topical sense, you can think about it as the NCAA
pools, starting tomorrow there'll be individual events,
games, which will lead to next events in the next round,
and next events in the next round, to an ultimate conclusion, in this
case, the NCAA champion, which can only occur if the proper
events have happened, prior to it.
And you'll see another specific example of how the order is assessed
in the development of the stages of the cell cycle.
OK, so we know that the cell cycle is important. The question is,
how is it controlled? How do you accomplish this orderly process of
cell cycle progression? What are the genes --
-- that regulate this process?
What do the genes encode, what do the proteins, and what do the
proteins do? What are the pathways that they regulate to ensure the
stages of the cell cycle? This was a huge question for
decades, and there was rather little progress in trying to understand how
it happened, especially in us. Our cells are sufficiently complex,
and there's relatively imprecise, or particularly was in the past,
imprecise methods for dissecting complex processes in
mammalian cells. And so it took experiments performed
in yeast, a single-cell, eukaryotic organism, budding yeast
and also fission yeast, to allow us to understand what the
details of the cell cycle are. Budding yeast, as shown here in a
picture from your book, is a single-cell organism.
It's also haploid, or it can be at least,
haploid, which means that it has a single set of chromosomes,
it doesn't have two sets, single set of genes, which makes it amenable to
genetic analysis. You can make mutants more easily,
if you only have one copy of each gene to worry about.
So that's another reason that yeast was attractive.
And another reason, which is kind of seen here,
not really obvious here, but when yeast cells divide,
they go through a very characteristic, morphological
change. They start off as spheres,
like this one, and as they go through the cell cycle,
they develop a small bud. That bud then gets bigger,
and eventually the two, the joint between the mother cell and the bud,
get severed to form two cells. And you can actually figure out where
you are in the cell cycle, by examining exactly what the
morphology of the yeast cell is, and I'll show you that on the next
slide. This is the yeast cell cycle,
with overlay of the diagram of what the cells look like in the different
phases, so a yeast cell that's in G0, let's say, looks like this,
fairly round, nondescript, as it enters G1 it creates a small bud,
that bud then gets bigger as the cells are now duplicating their DNA,
the bud actually gets bigger. It gets bigger still,
during G2 phase, and then at M-phase you can actually see a little
junction between the two cells, and the mother and the daughter cell
are more-or-less the same size, and then they separate from one
another, and they can go through the cycle again.
And this is useful because you can tell if you have a mutant,
as you'll see in a moment, if you have a mutant in a gene that affects
one of these processes, you can actually figure out where
the mutant gene acts within the cell cycle, based on what the yeast cell
looks like. And these individuals here, won the Nobel prize for their
efforts to understand the cell cycle, they won it about five
years ago, or so. This is Lee Hartwell,
he's an American, happens to run a cancer center out at the University
of Washington, very suave and sophisticated guy.
These two goofballs are Brits, very nice guys actually,
Paul Nurse, who's now the president of Rockefeller University in New
York, and Tim Hunt, actually Sir Tim Hunt,
because he's knighted after this Nobel Prize. And I'm going to
explain the experiments that all three of these guys did today,
which won them the Nobel prize, and gave us tremendous insight into
the control of the cell cycle in yeast, which turned out to tell us
how the cell cycle is controlled, also, in us.
OK, so the first thing we need to do is to create mutants.
That's a general theme in biology. If you want to understand a process,
find a mutant that can't do it, and understand the genes that get
mutated in that process, and so that's what happened in the
hands of Lee Hartwell, the guy in the upper-right,
he took this same yeast, the budding yeast,
which is also Brewer's yeast, the yeast that makes beer.
He took a population of these yeast
cells, and he mutagenized them.
I told you about this before, you can add chemical mutagens that
soak into the cells, bind to the DNA, cause mutations.
Each cell might have one, or a few nucleotides changed,
and at some frequency those mutations will affect genes,
and at some frequency the genes affected will influence the process
that you're trying to study. So he was trying to study growth
control, cell division, so he asked about the ability of the
cells to grow, and particularly,
he did so at two different temperatures. He tested the ability
of the cells to grow at their normal temperature, which is about 25
degrees centigrade, and he found that many of these
yeast cells would grow, following mutagenesis, at 25 degrees.
Probably some of them wouldn't grow
because they would've inactivated some critical gene,
and even at 25 degrees, they couldn't grow. However,
he then took these plates, and he used a technique,
which I referred to in a previous lecture, called replica plating.
He took a stamp, basically, of these colonies,
transferred them onto a fresh plate, and examined how the cells grew at
30 degrees centigrade. And what he found was that,
whereas many of the cells that were able to grow at 25 degrees continued
to grow at 30 degrees, so they produced colonies in exactly
the pattern that he had seen previously. Some of
the cells didn't. This colony here,
while it was successful, the cells were successful in growing
at 25 degrees, failed to grow at 30 degrees.
And this type of mutant is referred to as a temperature-sensitive
mutant, -- [PAUSE}
-- abbreviated TS. We imagine that this mutation
affects the amino acid sequence of the protein, at low temperatures the
protein is still able to function, but at higher temperatures, maybe it
becomes a little unstable, and now it can't function, and
that's why you see no growth here. So we had a TS-mutant that affected
the growth of the cells, OK? So my question to you is,
is that TS-mutant interesting? Is it interesting?
Well we can't actually know whether
it's interesting yet, because there are two general
classes of mutations that would give you this same phenotype.
One of them is interesting, does that cement always show,
or do we have a problem here? It's always there? Never noticed it,
in all these years. You can distinguish between these two
classes of boring and interesting mutants, based on the morphology of
the yeast cells when you shift the temperature. If you start with a
population of yeast cells growing just randomly at 25 degrees
centigrade, including these mutants, if you were to look under the
microscope, you would find mutants, cells that were at various stages of
the cell cycle. OK? Now, let's imagine that our mutation
affects a general enzyme that's required for cell viability,
DNA polymerase or maybe, better still, some ribosomal protein that
all cells need all the time, in order to function. If I were to
take these cells which were growing at 25 degrees centigrade,
and I were to shift them to 30 degrees centigrade,
and look under the microscope, what would I see?
Well, if it's a ribosomal protein,
and now translation just stops, these cells are going nowhere.
Regardless of where they were in the cell cycle,
they don't go any further because you need protein synthesis to do
anything, so if you look under the microscope at these cells,
following a temperature shift, you would still find a distribution
of cells in the different phases of the cell cycle.
Lee Hartwell was not interested in this class of mutants.
They could be anything, he didn't care. However,
he reasoned that if he were dealing with a mutant that affected
specifically a stage in the cell cycle, he might find that the cells,
upon temperature shift from 25 degrees to 30 degrees,
got to a particular point in the cell cycle and couldn't go any
further, that this gene affected one of these important transitions,
and so for example -- -- if he were to look under the
microscope, all of the cells would have arrested with a small bud,
would have arrested in G1, or maybe they would've arrested looking like
this, just prior to mitosis, or like this, somewhere in G2.
But importantly, they would arrest with a specific morphology that
would indicate that they had a specific cell cycle block.
And so he did this, and he found many, many such mutants.
He called them cell division cycle mutants, or CDC mutants.
And it was his methodology, which really broke this field open,
and won him the Nobel prize. Now one example of a CDC mutant that he
discovered, called CDC2, was particularly important.
It's been renamed CDK1, or cyclin-dependent kinase one,
for reasons that I'll come to. Cyclin, sorry, cyclin-dependent,
kinase one.
And this one acts at a particular phase in the cell cycle,
early on in the transition from G1 to S. So if you imagine cells in G0,
going to G1, and then progressing from G1 into S-phase,
into G2, and finally, in mitosis. This particular mutant blocked
right here, and so it said that this gene, CDC2 or CDK1,
was required for this transition.
And just to emphasize the TS-temperature shift and building up
of the cells in the cell cycle concept, imagine a cell that looked
like this, before the temperature shift. Ok? If you now do the
temperature shift, this cell will progress to this
point, but it won't go any further because CDC2 is required.
Maybe I'll draw this over here, to make it even more obvious.
CDC2 is required to go beyond this point, so that cell will arrest
right here. If there were a cell that looked like this,
in the population of 25 degrees, and now you shifted the temperature,
it would complete this phase because CDC2 is not required.
It would make it all the way to here, it would keep going,
and then it would get stuck here again. And that's why you get cells
building up with a particular morphology. OK?
So this was successful, and as I said, he isolated a large
number of such CDC mutants, which have taught us not about just
that transition, but many other transitions in the
cell. Now what Paul Nurse did,
this guy here, he actually was performing very similar experiments
in a related yeast species, but the critical experiment that he
did was to clone the gene that is responsible for CDC2 function.
So he took CDC2 mutant cells, which at 30 degrees --
-- will not grow.
And he added, through a cDNA library, CDC2 cDNA,
of yeast origin. So he made a cDNA library from yeast,
he introduced it into these mutant cells, and then he asked whether the
cells could now grow at 30 degrees --
-- and the answer was yes.
That is, if the cells now carried an extra copy of CDC2,
in the form of this cDNA, they now could grow at 30 degrees.
He complemented the mutation through the addition of a normal
copy of the CDC2 cDNA. Quite surprisingly, he did the same
experiment --
-- using not a yeast gene, but a human gene. Frankly,
there was great skepticism in the field about whether what these guys
were doing in yeast had anything to do with cell cycle control in humans.
Most people actually assumed that it probably had nothing to do with
it, and so therefore, when Paul Nurse proposed to try to
complement his yeast mutation with a human gene, people
were skeptical.
But in fact, it worked. And this told him, and the field,
that the machinery that controls the cell cycle in this simple,
single-celled eukaryote, is highly conserved, all the way through to
humans, and that we could therefore understand cell cycle control in us,
by doing experiments like this in yeast. So this really broke the
field open, and that's why Nurse won the Nobel prize at that same time.
However, there was a problem. They did in fact clone the gene,
they were able to sequence the gene, and they found that the sequence of
the gene, which I'm going to refer to now by the other name,
CDK1, looked like a kinase. It had amino acid sequence,
which made it resemble known kinases, so it was assumed to be a protein
kinase. However, it didn't have any kinase activity.
If you mixed it with various substrate molecules in the presence
of ATP, those substrates were not changed, they were not
phosphorylated. So the purified CDK enzyme had no
kinase activity, and therefore, it wasn't entirely
clear what its function really was, and the field sort of got stuck
there for a while, trying to understand the biochemical
function of CDK1, and other cell cycle regulators.
And that then led to the experiments done by Tim Hunt,
this guy here. And Tim Hunt, actually doing experiments at Woods
Hole, down at the Cape, in a summer course, with summer
students, did a famous experiment using sea urchins.
Sea urchins, when they're fertilized,
undergo very rapid, and very synchronous,
cell divisions. In the first few hours after you fertilize sea urchin
eggs, they will divide and divide again. And importantly,
they will do so in a very synchronous manner,
so all the cells will produce two cells at around the same time,
and those will all produce four cells at around the same time.
And this is important if one wants to do biochemical experiments,
to understand what is changing within these cells as they're going
through these various cell cycles. And so, what Tim and his students
did was to label the proteins in these dividing sea urchin cells,
with a radioactive amino acid, S35 methionine. And then they simply
made extracts of these cells at different time points thereafter,
and asked whether anything was changing in an interesting pattern,
at different times that correspond to different stages
of the cell cycle. So they ran protein gels,
they separated the proteins and then exposed the gels to x-ray film,
to visual what the protein concentrations were at different
time points. They took cells at time zero, they took cells after 30
minutes, after 60 minutes, after 90 minutes, 120 minutes,
150 minutes, 180 minutes. And they knew already,
from their earlier analysis, that this corresponded to the first
cell cycle, and this corresponded to the second cell cycle.
OK? Now some proteins, when they visualize them that way,
didn't change. At the different time points they saw roughly equal
concentrations of that protein throughout, but interestingly,
other proteins changed in abundance, and did so according to a pattern.
They seemed to oscillate, they seemed to cycle, in a pattern
that corresponded with the cell cycle. And so he called these
cyclins, and he suggested that they might have something to do with the
control of the cell division cycle. Well, meanwhile, Nurse and Hartwell
were doing their thing on CDKs, and so they came up with the idea
that maybe these two things have something to do with one another.
And particularly, maybe the failure of CDK to function
as a kinase was due to the fact that it didn't have an accessory protein
that it needed, mainly the cyclin.
And so, whereas CDK2, sorry, CDK1, was an inactive protein
kinase.
Now in a test tube, in a biochemical experiment,
if they mixed CDK1 with one of these cyclins, they observed kinase
activity. And thus the name, cyclin-dependent kinase. It's not a
kinase, it's not an active kinase in the absence of cyclin,
it only becomes active in the presence of cyclin.
So let me give you two quick examples of proteins that are then
phosphorylated by this active kinase, to give you a sense of how this
kinase regulates cell cycle transitions. There's a class of
proteins that are involved in the regulation of replication initiation.
We'll just call them replication initiation factors.
In the absence of phosphorylation, they're inactive, and that's one of
the reasons that replication is not initiated at those times
in the cell cycle. However, in the presence of CDK
cyclin, now the protein becomes phosphorylated,
and becomes active. OK? So one of the ways you trigger
the transition from G1 into S-phase, is to turn on this enzyme which
phosphorylated this target protein, and stimulates S-phase entry.
A second example is a protein called
pRB. In its un-phosphorylated state, it is active, different from here
where, in its un-phosphorylated state, it was inactive,
and the function of the RB protein, in its active state, is to block
again, the transition from S-phase to G1. This is actually an
important cancer gene, it's mutated in a large number of
cancers, and so we'll talk about its function, exactly how it blocks the
transition, in later lectures, but suffice it to say, it does that.
And when it is phosphorylated by CDK
cyclins, it becomes inactive, thereby allowing cells to progress
from G1 into S-phase. OK? So those are two examples of
how CDK cyclins operate.
Now, importantly, cyclin kinase activity is determined
by the level of the cyclin. As I told you, cyclin levels
oscillate, where this is cycle one, cycle two, cycle three. So these
are cyclin concentrations inside the cell. CDK levels do
not oscillate.
Concentration of the CDKs inside the cells is rather constant.
However, at this point in the cell cycle, when there's not enough
cyclin, you don't have kinase activity. Only when you go past the
threshold, do you get kinase activity.
When it drops again,
you lose kinase activity. But in the next cell cycle,
the cyclin levels increase again, and you get kinase activity, and so
on. So the oscillation of cyclins determines the oscillation of kinase
activity, which determines the periodicity of the cell cycle.
Now, for the truth in advertising, the situation is actually more
complex, there are actually multiple CDKs and multiple cyclins.
So in our cells, for example, if we imagine G1,
S, M, G2, G1, S, M, G2, there's a cyclin called cyclin-D,
which comes up in G1, goes down, comes up in the next G1, goes down.
There's another cyclin called
cyclin-E, which comes up a little bit later, in S-phase goes down,
stays down, comes up in the next S-phase. And there's finally
another one called cyclin-B, which comes up in G2, and comes down,
goes up in the next G2.
Anyway, the point is that there are different cyclins that get induced
in different phases of the cell cycle, and actually control,
through binding to different CDKs, different transitions in the
different phases of the cell cycle. OK. Let's see.
So, another concept: the transitions from the cell cycle don't occur,
from one cell cycle position to the next don't occur,
unless the previous cell cycle event has been completed.
And that makes sense because you don't want to,
for example, try to divide your DNA in mitosis if you haven't fully
replicated your DNA in S-phase. So there are processes that are
overlaid on top of cell cycle control, which ensure the completion
of one phase before the next phase is initiated. And I draw,
as an analogy, your washing machine, which likewise, has checkpoints
which will determine whether or not the previous phase of the wash cycle
has been completed. For example, you don't spin your
wash until all the water has been rinsed out. There's a sensor that
will determine whether that's not true, and if that sensor is tripped,
it blocks the wash cycle at that point. Your cells have very similar
checkpoints that will monitor and regulate cell cycle transitions,
and they're called checkpoints.
There are checkpoints, actually, that operate at different
phases of the cell cycle. I'll only give you one example,
it's actually the one that's best known. It occurs at the transition
in mitosis, from metaphase --
-- where, if you'll recall, the duplicated chromosomes line up
on the metaphase plate. In the process of anaphase,
where the chromosomes separate from one another, the chromatids I should
say, separate from one another. There's a cell cycle checkpoint
that makes sure that this happens before that can happen.
And in particular, if you have a chromosome which is
only attached to one side of the mitotic spindle,
so if you recall, these are centered in the middle
through attachment to microtubules that are emanating from the two
poles. If you have a chromosome that is only attached to one of the
two spindles, it's called monopolar attachment, whereas perhaps other
chromosomes, maybe all of the other chromosomes, are properly attached
at the metaphase plate. This one, unattached chromosome will
literally send a signal, a biochemical signal, which is the
equivalent of "wait for me". And that signal will inhibit cell
cycle progression. It'll specifically inhibit the
transition from metaphase to anaphase. While that signal is
being sent, the cells will just sit there, waiting for another
microtubule to bind to this end of the chromosome,
thereby extinguishing the signal, relieving this inhibition, and
allowing anaphase to progress. OK, so these checkpoints are
critical in insuring that these things happen in a timely and
ordered fashion. OK. That's it for the cell cycle,
so for the final ten minutes, and I always give short shrift to the next
topic, which is cell death, which is another fascinating topic,
fortunately there's not a lot of board work to show you.
Cell death. So cells are born, divide, as we've just been talking
about, but remarkable numbers of cells in your body die.
They will die, too, because of cellular injury,
but they'll also die because they're programmed to die,
or they'll decide to commit suicide, or they'll be murdered by other
cells. This happens in development, for example, your brains, as
developing fetuses, have ten times the number of cells
than you end up with as a young infant, because 90%,
for some of you more than 90% of the cells will actually be killed
through this process of programmed cell death.
Other cells, in your immune system, for example, are eliminated by this
process, so as to avoid them attacking your own body.
And there's many other examples of relevant and important programmed
cell death. So we want to understand this process,
partly because it's critical in normal development,
and also because deregulation of this process is critical
for many diseases. Too little cell death can give you
proliferate diseases like cancer or autoimmune disease,
too much cell death can give you diseases like neurodegenerative
diseases, too many cells in your brain dying. So the regulation of
cell death is quite important. Here are two other famous examples
of programmed cell death. This is the loss of the tadpole's
tail that occurs through an orderly, cellular suicide program, in which
all of these cells die, giving rise to a frog with no tail.
And likewise, in development, your hands are formed in such a way that
the digits are actually connected by other cells, but through this
process of programmed cell death, the cells in the middle, the
interdigital cells, are eliminated, thereby sculpting
the formation of your fingers. And this can be regulated,
and has been regulated, in evolution, in some species,
like ducks, the loss of the interdigital cells doesn't take
place, so they have webbing. In other species of birds, like in
us, the interdigital cells are removed, so they have sculpted toes.
This is what programmed cell death looks like. It is a very rapid,
and as I said, a very orderly process.
Cells get signals to die, they can get signals because they
get damaged, or they can get signals from their neighbors.
When they get those signals, they undergo a series of biochemical
changes. Their nucleus gets condensed, the DNA within the
nucleus breaks up, and then the cells themselves break
up into small fragments called apoptotic bodies,
and then interestingly, the cells that surround those cells,
eat the damage. It's like disposing of the body
after a crime, the cells are eliminated through a
process of phagocytosis, this is a very clean process then,
there's very little junk, there's very little cellular debris that
remains when this process of programmed cell death is completed.
This is a normal looking lymphocyte, and this is a lymphocyte undergoing
cell death. It's like, you know, this is your brain,
this is your brain on drugs, this is a cell, this is a cell undergoing
apoptosis. It's a fairly violent death,
at least visually. This movie shows you an example of that.
Here are cells undergoing apoptosis, I hope.
Maybe not. Oh well.
It comes from your book, so you can see it yourselves,
in fear that that would happen, I'll show you another set of stills.
Here are normal cells growing in the tissue culture dish,
you add some agent which induces apoptosis, the cells begin to round
up as you can see here, and their cell surfaces actually
become this horrible mess of blebbing membrane,
and they will eventually break up, as you can see starting to happen
here. So apoptosis is critical and
actually very, very interesting.
As I said, too much cell death can give rise to various diseases,
neurodegeneration stroke is complicated because of the effects
of apoptosis, too much apoptosis, even in AIDS, there's a lot of cell
death that takes place in apoptosis, and too little cell death, as I said,
is important in cancer, autoimmune disease, and certain
viral infections. Importantly, we know about the genes
that are regulated in cell death, that regulate cell death, through
genetic experiments. And here, the genetic experiments
were performed, in large part, at MIT,
by a professor in the biology department named Bob Horvitz.
He took comfort in the fact that the process in C.
elegans, a simple worm, has very many similarities to the
process that I've outlined to you, and therefore, he hoped that the
genes that regulate programmed cell death in C. elegans,
were similar to the ones that do so in mammals.
And so he then undertook a study to ask what genes regulate the cell
death process in this simple organism called C.
elegans, which as an adult has only 1,090 cells. And the beauty of this
organism is that you can actually see through it during development,
and you can therefore follow the fate of individual cells over the
course of development. And he and his students and fellows
did that, he observed that in the development of certain cell lineages
of the developing worm, individual cells died.
In fact, about 130 cells underwent this process of apoptosis.
And then he carried out a genetic experiment. He asked,
if I mutagenize worms, can I find ones in which this process doesn't
happen, or perhaps, in which this process happens too
much. What are the genes that regulate apoptosis?
And he found several such genes that turn viable cells into cells
that have undergone programmed cell death, or apoptosis.
In particular, there were two genes that positively
regulated this process. They were called Ced-3 and Ced-4.
They were required for apoptosis to occur. Mutants in Ced-3 and Ced-4
didn't have this cell death process in those developing worms.
And another gene called Ced-9 was required to prevent apoptosis.
In a Ced-9 mutant, there was too much apoptosis going on, OK?
They then cloned these genes, and it turned out they all have
homologs, versions, in our cells, and those genes in our
cells likewise regulate apoptosis in us. And we now know that at least
members of this family of genes are important in diseases,
including cancer. I'll just mention the function of one gene,
right now. Ced-3, it is a protease, which cleaves target proteins --
-- into fragments.
So, one of the key things that happens in apoptosis is,
you turn on this enzyme, this protease, and it goes around
cutting up various target proteins, which eventually lead to the death
of the cell. There are many target proteins that get cleaved when these
caspases, these proteases are called caspases, when these proteases get
activated, and I just gave you one example of one such target.
There's an enzyme that is normally involved in cleaving your DNA,
this is a DNA gel, of viable cells and cells that have undergone
apoptosis after a certain number of hours.
The DNA of the cells is normally intact, and large,
because this enzyme is kept in check. However, when its inhibitor is
cleaved by the caspsase, it now goes about cleaving the DNA,
and this is one of the reasons why apoptotic cells die,
because their DNA gets cut up into very small fragments.
There are a number of other targets, you can read a little bit more about
this in your book, I apologize that we had to rush
through apoptosis, it is important, and we will in fact
come back to it in a discussion of cancer.