Microbiology: RNA and DNA Tumor Virusespg. 1

Microbiology: RNA and DNA Tumor Virusespg. 1

Ryan Blankenship

Slide 2: We will all be impacted by cancer in our careers. Might be an oral cancer. Might be a Retinoblastoma. Lots of ways cancer will affect our professional careers. Shows us how viruses help us understand the progression of cancer. The goal today is to talk about how viruses in their normal mode of operation, take over cells in ways that are similar to what cancer does when it takes over the control of cells.

Review of cancer cells. They are unregulated, without any stops. All the mechanisms inside the cell that stop it from growing at inappropriate times are gone. They become immortal. The grow when they should be quite. They have increased nutritional uptake. If your going to grow you will need more nutrients.

When you grow them in culture they become what is called anchorage independent. Normal cells in our body have to be stuck to the extra cellular matrix. That part of what keeps them quiescent. Cancer cells lose the ability to need to be attached to the basement membrane and they do what is called anchorage independent growth. Then the cells grow at times when it is inappropriate for them to grow.

Gives two general example. One is the idea of cells becoming unregulated because signaling pathways become activated at the wrong time. A cell gets a signal to grow when it shouldn’t. The proteins that do that are called oncogenes. Oncogenes were first discovered in viruses called RNA tumor viruses.

The second is DNA tumor viruses. DNA tumor viruses have told us a lot about how cells shut off.

Cells normally have on the “brakes” but DNA tumor viruses have figured out ways to disrupt those brakes. The brakes inside the cell are called tumor suppressors and DNA tumor viruses have figured out ways to inactivate these tumor suppressors.

Slide 3: As you know all ready cancer cells grow in a lot of steps and it takes a lot genetic mutations to get to a full blown cancer. Lots of pathways gone wrong. An example is when a normal epithelial cell, you lose a gene, it starts to grow uncontrollable, another genetic mistake causes it to move further down the pathway towards cancer. This happens several times until it becomes a carcinoma and then with other mutations it begins to metastasize to other locations.

The development of cancer involves many steps each of which has to mutate the cell. Viruses have figured out how to act at these points to help themselves grow as viruses and shift the cell to a more cancer like state.

Slide 4: Types of cancer. RNA tumor viruses: leukemias (cancers that are derived from lymphoid cells,) carcinomas which are developed at the epithelial or endothelial cells, and sarcomas which are derived from connective tissues. DNA tumor viruses: all three kinds.

Slide 5: Key concept for the test: what is the difference between an oncogene and a tumor suppressor.

An oncogene is what geneticist call a gain of function mutation. It’s an altered gene product that can act in a dominant fashion that makes a cell act more like a cancer. Oncogenes were found in RNA viruses and they were found to be mutated form of a normal gene called a Protooncogene. Often associated with control of cell signaling. The converse of that are tumor suppressor genes. These are called a loss of function mutation. It is like a brake. It’s normal activity prevents formation of a tumor. When you lose that function the cell now loses the ability to stop itself from growing and becomes cancerous. One is a gain of function the other is a loss of function. RNA tumor viruses usually have oncogenes in them and DNA tumor viruses generally inactivate tumor suppressor genes.

Slide 6: One famous oncogene is called Myc. When myc is over expressed in a transgenic mouse you see that over time the percentage of tumor free mice go down. Ras is another important oncogene. The same thing happens when you put this into a transgenic mouse, but it happens more quickly than Myc. If you put the two of them together in the same mouse the rate increases again. They are able to together to cause the cells to move further along towards being a cancer.

Slide 7: Oncogenes are dominant mutations, gain of function, over expression mutations. If you do that with only one copy of the gene it’s enough to turn the cell into a cancer. Tumor suppressor genes normally keep things shut off. When you lose that shut down function you move to a cancer but you have to shut both copies of it. One copy is sufficient to keep things under control. DNA tumor viruses don’t work to mutate the genes but work to over come the effects of the tumor suppressor proteins.

Dominant mutation, only one copy of the mutation needed to cause the cancer. Tumor suppressor you need to knock out both copies to start the cancer.

Slide 8: Don’t need to memorize this slide. The point of it is to show us all the places that there are oncogens and tumor suppressors in all of these pathways which control the expression of genes in a cell. The red dots are where known oncogenes and tumor suppressors that in normal times keep things under control. When you have oncogenes or tumor suppressor inactivation can cause it to shift towards being more cancerous. The point of this is to appreciate that there are many many place where these control circuits can go out of whack and cause a cancer.

Slide 9: There are two kinds of tumor viruses, RNA and DNA.

Rous sarcoma virus (RSV) is a RNA virus that causes a sarcoma in chickens. Forms solid tumors. This is named after Peyton Rous. What he discovered in 1911 were tumors that were found in chickens and if you cut that tumor up and ground it up into liquid and pulp and filtered it so only the liquid came through, the filtered liquid was capable of transmitting the disease. Since it was filterable it wasn’t bacterial. It wasn’t pieces of the tumor causing the disease. It was a virus. He discovered temperature sensitive copies of the mutants of RSV. This means that it does something at one temperature but not at another. A chicken is normally at 41 degrees Centigrade body temp. Rous found a variation of the RSV virus that grew at 37 degrees Centigrade. At 41 degrees in a chicken RSV made the virus but no tumors. At 37 degrees there is a specific protein that still works in the virus, but at 41 degrees that protein is unfolded and doesn’t work anymore. Also you can conclude that whatever is causing the formation of tumors in the virus, resides in a protein the virus makes. Rous had the first evidence for what we eventually defined as an oncogene. At 37 degrees RSV would make tumors but not at 41 degrees. That’s part one of the story.

Part two of the story is Peter Vout. He discovered that you could take mutants of RSV and among them you could find viruses that no longer make tumors at 41 degrees. They were deletion mutants not temperature sensitive mutants. What that means that a piece of the RNA genome in the virus is missing. Compared to the one that can cause tumors. That piece of genomic information was called sarc because it was the thing that caused these sarcomas. What ever was deleted was sarc.

Part three of the story is Herald Varmos and Mike Bishop. They won the Nobel prize for a really simple experiment. You have RSV that can make tumors. You got deletions in another RSV which cannot make tumors. Reverse transcriptase is the conversion of RNA to DNA. Found in any retrovirus such as HIV. In the early days of understanding reverse transcriptase, they couldn’t make very long copies of DNA. What they did is they took the RSV which makes tumors and contains the sarc gene and used reverse transcriptase and made a lot of DNA copies of the RSV’s genome and its sarc gene. Then they took these copies and hybridized them to copies of the RNA of the RSV that had the sarc deletion and all of the DNA copies minus the sarc copies annealed to the deletion RSV’s RNA. This left over a pot of sarc. What won them the Nobel Prize was taking the left over copies of the sarc gene DNA and annealing it to chromosomal DNA. They discovered that 60% of the DNA copies annealed to the chromosomes. So what ever the sarc gene was it was found in the chromosomal DNA. This lead to the idea of a protooncogene. A normal cellular copy goes bad, which mutates it, that does something to the gene and now converts the cell to cancer. The protooncogene goes out of control and converts to cancer. That’s the history of RNA tumor viruses. There are other RNA viruses that are thought to have some involvement in human tumor cells but we don’t know exalty how yet.

There is HTLB 1 which is a human T Cell leukemia which is what people originally thought HIV was. About 1% infect will eventually develop a cancer. HCV is an RNA virus that is associated with hepatocellular carcinoma.

There are also DNA tumor viruses. Guardasil is a vaccine that is specific HPV that is associated with cervical cancer. Burkett’s lymphoma is caused by a herpes virus, both the EBV. It can lead to an immunological defect that causes a tumor or cancer to occur. HBV is a DNA virus also associated with carcinoma.

Slide 10: RNA tumor viruses we think about in two ways, one is acute transforming viruses which means that they can transform cells in tissue culture. RSV is replication competent but many RNA tumors viruses are replication defective. They carry oncogenes which can in a short period of time cause tumors. The non acute viruses don’t carry the oncogenes, can’t transform cells in culture but can cause tumors in animals over a very extended period of time.

Slide 12: Understanding the oncogene. This covers what he covered before about Rous, Vout, Varmous and Bishop.

Slide 13: Here are examples of RNA tumor viruses. Here is RSV, the sarc oncogene is down at the very end and it make a protein called P60 sarc that causes the tumor. This virus is replication competent. There are many that carry other oncogenes like Myb, X, Erb a and Erb b that are replication defective. You have to a mixture of wild type and this virus to be able to grow in the wild. It is genetic changes. We have talked about oncogenes and protooncogenes being in our chromosomes. It takes genetic changes to make that protooncogenes into an oncogene. Two examples. Epidermal growth factor receptor, normally it responds to epidermal growth factor. In the Erb b oncogene, the stuff out here that senses the EGF is missing. When it is missing this gene thinks it’s there. It’s on all the time. It’s sending a growth signal all the time. Here is the new oncogene. With a particular mutation it doesn’t matter what’s on the outside of the cell, it always thinks it’s on. Sends a growth signal at the wrong time. All of these oncogenes are part of growth signaling pathways which tell the cell it’s time to start growing.

Slide 15: For the purpose of the test you need to know all four kinds of oncogenes and an example of each.

Slide 16 and 17: The first kind of oncogene is one that acts like a growth factor. A virus carries this oncogene, it makes its protein, the cell thinks that the growth factor is out there and its binds its receptor and it turns the cell on. These are very rare. Only two. Remember the Sis oncogene and you’ll be fine. It is a phony growth factor. Sis floats around like a growth factor looking for its receptor.

Slide 18 and 19: The second kind of oncogene is called mutated receptors. These are receptors that tell the cell its time to grow whether or not the signal is there. There is Erb b, new, fms, ros and sea are all receptors that are mutated and always tell the cell it’s time to grow whether it is or not.

Slide 20 and 21: The third kind of oncogenes are called intracellular transducers. There are four kinds of this particular class: The tyrosine-kinase, the seronine threonine kinase, the Ras protein, and the phosphokinase C. Four different kinds of proteins that send signals to the nucleus that it’s time to grow when it’s not time to grow.

Slide 22: The fourth kind are actual transcription factors. Jun and Fos were the first to be discovered because they are the two subunits of the transcription factor of many genes called AP 1. Myc and Myb and Rel and Erb a are all examples of transcription factors that can work as oncogenes when they are mutated.

Slide 23: You can find the oncogenes in more than one virus. Erb can be found in three different viruses and myc can be found in six different kinds of viruses. It easy to get these oncogenes encoded in different viruses in different organisms. He shows examples of myc oncogene variations. One of these is found in cat and the other in chicken.

Slide 24-26: Up to now has been acute transferring viruses. These happen quickly, one to two months. If the oncogene gets activated the tumor is detected in one to two months. The chronic viruses are the non acute viruses that don’t carry oncogenes and act at a distance. It’s what we call oncogenesis by insertion. What it does is that it mucks up the normal control of the protooncogene. It turns the protooncogene on when it shouldn’t. It takes a really long time, one to two years. They work by insertional mutagenesis. They insert somewhere close by and turn on protooncogenes at a time when it’s not suppose to be on. Sometimes they do that by chromosomal rearrangement. They’ll insert and cause regulatory signals to be rearranged or they’ll use their own promoters to turn on the expression of genes when it shouldn’t. They over produce the protein at the wrong time. They work slowly but they are potent tumor viruses. You put the virus in here. It’s usually a retrovirus. It’s promoter turns on the nearby protooncogene. You might actually fuse the promoter itself to the oncogene and turn it on. You might cause it’s RNA splicing to be different. Or you might turn off a gene and that acts more like a tumor suppressing gene problem. They don’t carry an oncogene like the acute RNA tumor viruses, they do something that alters the expression of a protooncogene so it works at the wrong time.

Slide 27: Acute viruses work quickly and carry there oncogenes generally and can see tumor in a month or two. Chronic viruses don’t carry oncogenes, and they work by inserting somewhere close to a protooncogene and turning it on at a time and place that is inappropriate to keep it from growing.

Slide 28: Comparing this to DNA tumor viruses. Oncogenes and growth signaling pathways, which is telling the cell to grow when it’s not time to grow are attributes of RNA tumor viruses. DNA tumor viruses tell the cell to take the brakes off the cell. The cell have signals that tell it to grow and to stay off. DNA tumor viruses take away the signal that tell it not to grow. The human adenoviruses, papilloma viruses, which cause cervical cancer by taking the brakes off. Then there are also parpovaviruses like SV 40, JC and BK viruses, which all cut the brake line.

Slide 29: In each case these DNA tumor viruses have proteins in them that bind to the tumor suppressor proteins like pRb and p107 and p130 and p53. Each of these make a protein. In the case of adenovirus its E1A, in the case of papilloma virus its E7, in the case of SV 40 its large T antigen that will bind and muck up the brakes.

Slide 30: Retinoblastoma is an ocular tumor that occurs in about 1:20,000. There are two forms the hereditary form and the non hereditary form. It’s a deletion in a chromosome. It’s recessive. It’s likely to be a DNA tumor virus. That’s because when this gene is knocked out in both copies of the chromosome 13 you form the ocular tumor. Retinoblastoma is a tumor suppressor when it’s working.

Slide 31, 32, 33, 35: The gene product of RB is an anti proliferation protein. It is a tumor suppressor. Not only is it found in the retina but is found in breast cancers, in small cell lung cancers. It is an important target for how the cancer gets started. It regulates genes that are involved in cell growth. Particularly in genes that regulate DNA replication. You can overcome the cancer phenotype in cells by putting in copies of the clone RB gene. It restores the tumor suppressor mechanism. DNA tumor viruses act by interfering with the way RB normally acts.

RB normally binds to a transcription factor called E2F. E2F controls cells DNA synthesis. In a normal cell, RB inactivates E2F so that DNA synthesis genes can’t be expressed. Until the right time of the cell cycle when RB is phosphorylated and lets go of E2F. Now E2F can make all the things that are required for DNA synthesis. In G1 RB is inactive, in S it reasserts its binding to E2F so that no more DNA replication proteins can be made. RB is only inactivated during the G1 phase and a little bit of the S phase. Then it is reestablished to be able to bind E2F.