Thursday, March 26, 2009
- Today we’re going to discuss cancer & as we can see from these
statistics, this is a major issue in society to deal with.
- The key thing about this slide is that these pathways are also the
pathways that are critical for normal cell biology so the same pathways
that are involved in normal cell biology are also involved in cancer. They
somehow go awry & can lead to cancer.
- So we can see for example here in this part, we’ve got cadherin
integrins, we can see cell-cell interactions can be implicated in cancer.
- Here we can see pathways downstream of growth factors or mitogens
that will lead to the activation of Myc & deriving progression through the
- Here we have control of cell death.
- Here we have sensors of DNA damage and p53.
- Here we have another pathway related to the cell cycle.
- So these same pathways that control normal cell biology are implicated
~10 cells in the human body
One of these cells
- So there are a lot of cells in the human body, there are 10 cells in the
human body and only one of these can lead to the development of cancer.
All of these cells here, they are experiencing mutations everyday, we’ve
got billions of cells in our body that are experiencing mutations. Many of
these mutations are being fixed by the DNA repair machinery that is in all
of our cells so most of those mutations will be fixed right away. Those
that can’t repair their DNA damage, the body will destroy those aberrant
cells as we learned about with p53 inducing apoptosis if there is
persistent DNA damage.
- But cancer can develop from one of these cells if they can gain a
mutation that allows it to survive & divide, therefore forming a tumour.
So if it can escape these normal repair mechanisms, these normal
apoptotic mechanisms that would destroy aberrant cells, cancer can
develop. The cancer will develop from one single cell – it’s a clonal
process, it grows from one single cell dividing & evidence for that is
shown in the next slide. - Here is evidence for the clonal origin of a tumour.
- And you can see in this illustration right here, b/c we see 2 different cell
types shown in this tissue right here. Now these 2 different cell types
shown here in red and gray they represent cells that have one X
chromosome inactivated or another X chromosome inactivated.
- So females have 2 X chromosomes to match the gene expression that
occurs in males so that we all have the same gene expression on our sex
chromosomes. Males have an X & a Y, females have an X & an
inactivated X & that evens out the gene expression. So one of the 2 of X
chromosomes is inactivated in female cells & this is for this dosage
compensation so that we all have the same gene dosage from the X
chromosome. But this inactivation occurs randomly – one of the X’s
could be inactivated or the other one could.
- B/c there is a random process of inactivation, these cells will inactivate
one X or the other, so we see a red cell or a gray cell.
- Now if you’ve analyzed the cancer in this tissue, all of these would have
exactly the same X chromosome inactivated. So what this means is the
tumour was derived from a single founder cell – one of these single red
cells in the picture.
- If it was founded from a group of cells as seen in the blue circle, then
the tumour would also be a mixture of these 2 different cell types, the red
& the gray.
- So these sort of experiments here show that they’re all being derived
from one cell type in this tissue. So this is one example which shows the
clonal origin of this tumour – tumours can arise from one single cell.
- But even though they can arise from a single cell, this is very dangerous
as illustrated right here b/c when a single cell divides, this is an example
of exponential growth so one cell will reproduce 2, 2 will produce 4 & 8
& so on.
- Diagram: Here are the #s of population doublings across the bottom of
this graph & you can see by the time this single cell that’s started out
here, by the time it’s doubled 30 times the tumour size is approaching a
centimeter across. Here when it’s a centimeter across, this is a size that is
palpable, it can be detected by a physical exam by a physician, if it
undergoes 10 more doublings, now this tumour will be 10 cm across so
it’s going from a size like this then in 10 doublings, it will be 10 cm
across & this size of a tumour is going to be deadly for the patient.
Uncontrolled growing mass of abnormal cells
- So let’s just go through some of the categorization of different types of
tumours. These are all different types of tumours, & they’re classified
based on the cell of where the cell originated from in the body that led to
- In addition to tumours being classified based on their origin in the body,
they can also be classified based on their severity. Invaded surrounding tissue
- Here we’ve got first off, we have a benign tumour & here’s a malignant
- So this benign tumour, this is a growing mass of self-contained cells,
it’s a single tumour. So here this one would have been arising from an
epithelial origin. Here you see the epithelial cells here, here they’re
starting to divide uncontrollably but notice that this tumour is surrounded
by the basal lamina – it’s contained by the extracellular matrix so it’s a
contained unit, it’s growing but it’s contained in the body.
- The difference then going to a malignant tumour is that has become a
more aggressive tumor b/c it has broken free of this basal lamina and it
starts to invade the surrounding tissue.
- So both tumours here are growing, the cells continue to divide. In this
case (benign), it’s contained within the basal lamina, contained within
one spot in the body & here (malignant) the cells are still dividing but
now they’re escaping that initial position in the body, leaving & invading
- Now the next step after this is even more dangerous b/c now we have a
metatastic tumour. Now the tumour not only invades the surrounding
tissue, but it can invade tissues all through the body.
- So here up at the top we’d have our normal epithelium with the basal
lamina right below it. Here we’d initially have a benign tumour starting to
grow so it’s self-contained, it’s kept in this part of the body b/c of this
- Now it starts to become malignant so it’s leaving that initial site,
breaking through that basal lamina. Once it does that, it can start
migrating through the extracellular matrix, the connective tissue & reach
another compartment of the body, either the blood vessels or the vessels
of the lymphatic system & these cells can travel through the lumen of
vessels to completely different sites of the body & then leave these blood
vessels to then form a secondary tumour in another part of the body. So
this is the most dangerous type of tumour where you have the tumour
cells spreading throughout the body.
- Now the key thing for all of these cancers is that cancer cells have
- So in this slide here, this is called a karyotype & what it is is all of the
chromosomes in a cell laid out on a glass slide & the chromosomes have
been stained with different dyes. So here, this is a normal array of st
chromosomes for a human cell so the yellow stain marks the 1
chromosome, this brown stain the 2 & the gray the 3 & so on.
- So you can see in a normal cell, we have 2 of each chromosomes b/c
we’re diploid organisms. Have a look at this cancerous cell – this is a
very advanced cancer cell, there are multiple disruptions in this genome –
there is not just a single disruption that would start the process but it has
accumulated more & more genetic instability as it’s developed. You can
see in this one cell, there are now 4 1 chromosomes. here we have 6 2 nd
chromosomes, so this cell is in big trouble – it has many extra copies of
these chromosomes – this relates back to cell division & their proper
segregation to the 2 daughter cells & if that doesn’t happen properly, then
one daughter cell can start to accumulate extra chromosomes & this is
what we can see in here in this cancer.
- In addition to the additional numbers of chromosomes, we can also see
chromosomes that have multiple colours too. So what does that mean?
Here we’ve got the number 10 chromosome with a purple bit on it – the purple actually labels the 12 chromosome normally. Here we have the 12
chromosome & the 10 chromosome separate so that means that a piece of
this 12 chromosome has translocated and attached itself to the 10
chromosome so this is another major rearrangement of the DNA, a
disruption to the normal DNA in a cancer cell.
- So there are mutations then that cause cancer.
- So first off we’re going to discuss what causes the mutations that lead to
cancer. Note down that these are mutagens, these are anything that can
cause mutations in any organism. These could be chemical mutagens,
they can be radiation, they can be viruses. These are all things that can
cause changes to the DNA sequence or the DNA structure, they are
- Here is an example of a chemical mutagen.
- Here is this aflatoxin which is a chemical that is found on mould,
growing on peanuts & if you ingest this aflatoxin, in our bodies, there is
this enzyme here, cytochrome P-450, this converts the aflatoxin into this
secondary product. This secondary product here covalently attaches itself
to guanine in DNA. So here is our DNA in the nucleus of the cell, now
we have this chemical attachment onto our guanine.
- Notice here that this looks an awful lot like a DNA base so by attaching
this chemical on here, this will insert into the other side of the double
helix of the DNA & so instead of this guanine binding to a cytosine on
the other side of the DNA, that cytosine will now be lost & this will take
its place so we will delete a cytosine from the DNA, we create mutation
in DNA b/c of this covalent attachment onto the guanine.
- So this is one example of a chemical mutagen, there are many of them,
so how can we detect whether a substance can mutate DNA? And to do
this you can use what’s called the Ames Test.
The Ames Test
- Now he wants to emphasize right away b/c there’s often confusion
about this test: this test is looking for the ability of a chemical to cause a
mutation, just that, only that. It’s not asking whether a chemical can cause
cancer or anything like that, just can a chemical cause a mutation? And
specifically, we’re asking whether a chemical can cause a mutation in this
Salmonella bacteria. This test uses a specific type of Salmonella, a strain
that has a defect in a gene for histidine synthesis so this starting culture is
dependent on outside supply of histidine, it can’t synthesize it itself,
there’s a defect in the gene for histidine synthesis.
- And what we’re asking here: If we add our potential mutagen to these
bacteria, will this cause a mutation in that broken histidine gene & revert
it back to normal? Normally, we think of mutation of being a bad thing,
you create a mutation in a gene & it breaks it; this is a mutation that
causes a reversion, a reversion from a broken gene to a functional gene.
That is the basic thing that this assay is testing.
- So if we walk through this: If we imagine there is no mutagen here at
all, we just mix this histidine dependent Salmonella with water so we mix
it with water & we plate it out on a plate that has no histidine – there is no
histidine on this plate at all, this Salmonella depends on outside histidine
so no Salmonella will grow, very few of the Salmonella will grow.
- Now if instead we added a chemical mutagen here, this will lead to
mutations across the genome of this Salmonella & some of those
mutations will fix this broken His synthesis gene so if we take these
bacteria, plate them out on media that doesn’t have His, now many of them will grow b/c they now have this repaired histidine synthesis gene.
- So you can then run through a whole array of chemicals & ask can these
chemicals induce this mutation that reverts this histidine synthesis gene
back to normal & test whether you get growth on colonies afterwards so
it’s changing these histidine dependent Salmonella into histidine
independent Salmonella at the end.
- So that’s the basic idea of this test. One thing here, so why would we be
adding this homogenized liver extract to this assay? If we go back one
slide, remember here that this molecule here, this one that would be the
one that would be ingested from these moldy peanuts, but it doesn’t
directly bind to the guanine in the DNA, there is an enzyme in the body
that converts it to a second product that does that. So here you need this
enzyme to actually create the mutagen & that’s what this liver extract
supplies so this liver extract here is basically a cellular extract containing
all sorts of enzymes normally found in cells so if the primary chemical
doesn’t cause the effect, you could also have these secondary products
made in this reaction & test if they do.
- So these are the sorts of things, chemicals, radiation & viruses, they can
cause mutations but which mutations actually lead to cancer? What are
the genes that are mutated?
Gain of function mutation
Loss of function mutation
- So there are 2 main types of genes associated with cancer. One class is
oncogenes & the other class is tumour suppressor genes.
- Oncogenes – Proto-oncogene are normally proteins that are involved
driving the cell cycle forward. These are go-signals for the cell cycle,
they tell cells to divide in the normal process. The problem here is that
they become overactive, there is a gain of function, too much function so
now you have too much activation of the cell cycle & that can lead to
cancer so that’s called an oncogene.
- Tumor suppressor genes – These are genes that normally protect the
cells, that normally stop the cell cycle so now with this loss of function,
we’re losing stop signals for the cell cycle. We’re losing that inhibition of
the cell cycle & now the cells will tend to divide more & again, that leads
- So either we have hyperactivation of go-signals or we’re losing stop
signals & both types of these mutations aberrantly enhance cell
proliferation & survival.
- So we have to remember that human cells are diploid so we have to
consider whether these mutations are dominant or recessive if one of
these mutations occurs in the body.
- Diagram: Here is an over-activity mutation, a gain of function mutation,
creating an oncogene so here we have the mutation occurring so this gene
now is hyperactive – it’s producing way more protein activity than
normal. So it really doesn’t matter what’s happening on the other
chromosome, the gene on the other chromosome is producing the normal
activity of this gene but the oncogene, this mutated gene is the one that is
overactive so this normal gene can’t do anything to counter this, there’s
just elevated levels of either Ras or Myc in this cell that will drive the cell cycle forward.
- Now the opposite is true for tumour suppressors so they are recessive.
So here in an under-activity mutation, a loss of gene function, here if we
have a mutational event that inactivates a tumour suppressor gene, so now
this one has been inactivated so this one stop signal is lost, but there’s a
stop signal being produced from the other chromosome. So this cell will
still be able to control its cell cycle b/c there’s still an intact stop signal
that will regulate the cell cycle – it’s only when you lose both copies of
this, mutate both copies, that then you’re in danger of developing cancer.
- So up here (oncogene) it’s dominant, you only need one copy to be
mutated. Down here (tumour suppressor) it’s recessive b/c if you have
one copy mutated, the other one can still function & you require both to
be mutated to have a problem with the cell cycle.
- So how are these mutations actually generated in the body more
- 1 let’s go through gain-of-function oncogenes. Here we have mutations
that are leading to hyperactivity of the proteins expressed by these genes.
So in each of these cases, we have excess activity that’s pushing/driving
cell proliferation, driving overgrowth of this tumour. So this can occur for
example here, it could be a deletion or a point mutation in the coding
sequence, just a single base pair change could cause there to be a
hyperactive protein made, but this protein would be made in normal
- We’ve learned about a lot of proteins that could either be on or off. One
example of this would be a Ras which is a small GTPase. In the GDP
bound state, it’s off; when it binds GTP, it’s on & it activates Myc & the
cell cycle. So if there is a single mutation that causes that Ras to just
constantly bind GTP, it will be constantly on,