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BIO241 Lecture 22

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Jennifer Harris

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 cell cycle. - 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 in cancer. 14  ~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.  (Exponential growth) - 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 this cancer. - 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 tumour here. - 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 surrounding tissues. - 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 basal lamina. - 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 disrupted DNA. - 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 mutagens. - 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 to cancer. - 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 specifically? - 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 amounts. - 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,
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