Fundamentals II: 11:00 - 12:00 Scribe: Christopher Bannon

Fundamentals II: 11:00 - 12:00 Scribe: Christopher Bannon Tuesday, November 9, 2009 Proof: Brittany Paige Paugh Dr. Engler Tumor Viruses Page 1 of 5 TSG = Tumor Suppressor Gene, RSV – Rous Sarcoma Virus, GF – Growth Factor, Rb – Retinoblastoma, AA- Amino Acids

I. [S1]: How RNA and DNA viruses help us understand oncogenes and tumor suppressors a. Virus is a model system for how we understand cells. II. [S2] Characteristics of Cancer Cells a. How the use of viruses helps our understanding of how cancer cells work. We have learned a great deal about cancer through studying tumor viruses. We’ll talk about RNA tumor viruses and DNA tumor viruses and how they lead people to win Nobel prizes and things like that. III. [S3] Class Discussion Question a. According to what you know about cancer, is there a difference between the top 3 cancer types most likely to be diagnosed in 2008 in men and women, according to the American Cancer Society? i. Men = Prostate, Lung, Melanoma (reality Prostate = 25%, Lung = 15%, Colon = 10%) ii. Women = Breast, cervical, (Reality Breast = 26%, lung = 14%, Colon = 10%) b. Is there a difference in case number between 2007 and 2008? i. For men the total number of diagnosed cases of cancer dropped 20k from 2007 and for women it rose 14k from 2007 1. Regular tests/check-ups help the diagnosis (PSA tests for men, mammograms for women, dental oral exams, look for retinoblastoma for optometry, etc.) 2. It varies from year to year IV. Characteristics of Cancer Cells a. Tumors begin as cancer cells that won’t “shut off”, undergoing unregulated growth (immortal) they won’t shut off b. Oncogenes start cells growing and tumor suppressor genes are the one s that put the brakes on c. There is increased nutrient uptake and the cells become anchorage independent d. The main idea is that the cell cycle becomes active when it shouldn’t e. RNA tumor viruses (where concept of oncogenes originated) i. Oncogenes are growth-signaling pathways, which are active at the wrong time  tumor. At the right time would be in times of wound repair and things like that f. DNA Tumor suppressors (1st Identified in DNA tumor viruses) i. These are pathways in cell that prevent cell proliferation. They keep the brakes on the cell to keep it from growing g. The fundamental lesson today is that Oncogenes promote increase nutrient uptake and promote rapid cell (give it gas in terms of the car analogy) growth in an unregulated fashion, where as tumor suppressors keep pathways turned off (get deleted/ inactivated in cancerous tissue removing inhibition) V. [S3] Cancers develop in many steps due to the many mutation events a. Multiple steps/events that “go wrong” in cell before a tumor develops. You can’t predict necessarily what the tumor i. Genetic and other events (mutations/ deletions)  convert healthy cell into a tumor cell/ carcinoma allowing metastasis other sites in the body VI. [S4] Some types of cancer associated with tumor viruses a. There are specific kinds of tumors the 3 most often diseases associated with tumor viruses i. Leukemia = cancer of blood (avian leukemia virus) ii. Carcinoma iii. Sarcoma (ex. rouse sarcoma virus) VII. [S5] Definitions a. Oncogenes = gain of function mutation, i. Gene product is either made at wrong time or made in way that acts in a dominant fashion (this is something that controls phenotype), usually mutation of normal proto-oncogene which controls normal cell growth and division. It gains a function. It makes thing s happen in the cell that shouldn’t happen at that time b. The other kind of mutation is the tumor Suppressor gene mutation: tumors resulting from this are caused by a “loss of function”; its normal activity prevents formation of a cancer and this loss of activity allows a cancer to develop b/c of the mutation. The brakes are now off. VIII. [S6] Oncogenes can increase tumor susceptibility in transgenic mice a. Here’s an example of Oncogenes that are inserted into transgenic mice. You are going to ask yourself how many mice remains tumor free after so many days. So if you place Myc Oncogenes into transgenic mice, allowing it to be expressed  you don’t see tumors for several days then the number of tumors increase and the number of tumor free mice decreases Fundamentals II: 11:00 - 12:00 Scribe: Christopher Bannon Tuesday, November 9, 2009 Proof: Brittany Paige Paugh Dr. Engler Tumor Viruses Page 2 of 5 b. Ras oncogenes = another oncogene that was identified from viruses. It poses an earlier onset of this formation of tumors and the rate at which the tumors are found increases (compared to Myc) c. Then if you have a Transgenic mouse with both Myc and Ras you’ll see that the rate increases even more these two mutations work synergistically for tumor development. i. Accumulation of these types of mutations in a cell that is on path to become cancerous  speed up rate of tumor development IX. [S7] How do oncogenes and tumor suppressors work? a. TSG = gain of function mutation, and if it occurs in even 1 place of chromosome = start down path of cancer development b. Tumor suppressor you need to lose a function. And since you’re chromosomes have 2 copies of all tumor suppressor genes you’ve got to mutate both copies of the TSG in order for cancer to develop. (Knock out both sets of brakes) we’ll see late on that DNA tumor viruses inactivate both copies of this gene by sucking up all of the protein that these genes “make” so that it can’t carry out its intended function. X. [S8] There are many pathways affected by oncogenes and tumor suppressor proteins a. Don’t memorize, just showing all the places where Oncogenes or tumor suppressor genes have been indentified. All these red dots are potential places where you’re cells could go bad and make a tumor b. Mutation in many sites, such as expression of an oncogenes (Ras and Myc) or the removal of cell control (as with Retinoblastoma or p53). c. Any of these mutations can lead to tumor development; there are lots of vulnerable sections in the genome to mutations XI. [S9] General overview of DNA and RNA transforming viruses a. Viruses can be isolated and purified so they are an important genetic tool. They can infect cells and many times they take advantage of normal cell function to replicate more viruses b. Some viruses naturally cause tumors i. Rous sarcoma causes solid tumors in chicken c. Viruses can also cause tumors indirectly (DNA viruses) i. Cervical cancer (HPV associated with cervical cancer). People who have received the Gardasil vaccine have developed immunity to HPV to try to stop association of virus with formation of cervical cancer ii. Epstein Bar virus is involved formation of mononucleosis/ kissing disease 1. Can lead to Burkitts Lymphoma, a cancer of the of nasopharyngeal cavity (immunocompromised people) it can be deadly iii. Hep. B = involved in liver cancer. iv. Those are the DNA viruses associated with cancer d. RNA viruses are also associated with cancer i. HTLV-1 = T-cell leukemia (1% of those affected will eventually develop a cancer) it’s a cancer of the blood cells ii. Hepatitis C = involved in liver cancer (RNA virus not a DNA virus) iii. So RNA viruses have a long history of being involved in the cause of cancerous tumors XII. [S10] Retroviruses life cycle requires integration into the chromosome a. Retroviruses, upon infection of host cell, use reverse transcriptase to make a DNA copy which is then inserted in to host chromosome i. Location of the insertion into the chromosome can disrupt nearby genes leading to cancer development XIII.[S11] Retroviruses can cause tumors in two ways: a. Acute Transforming Viruses = carry oncogenes in their own viral genome and are able to replicate by themselves or be defective in replication and require helper virus b. Chronic/ Non-Acute = do NOT carry oncogenes but can often replicate and can form tumors, but much slower i. Done through insertion into chromosome and disrupting the regulation of genes around the site of insertion. ii. So these need a long time to get started but they can eventually cause tumor cells. We’re going to start off by talking about the RNA viruses that do have Oncogenes XIV. [S12] Chronology of understanding oncogenes a. Who’s interested in winning a Nobel Prize? i. Rous Sarcoma Virus (virus that affects chickens) probably one of the earliest viruses that were ever characterized 1. Took tumors out of chicken and removed cells from tumors and ground them up in saline and transplanted the extraction into other chickens  all the chickens injected with the extract developed tumors (caused by virus) 2. Temperature sensitive mutant = virus grows at 1 temperature but not at a higher temperature a. Some Rous sarcoma viruses that made tumors at 37 deg C but not at 41 deg. Fundamentals II: 11:00 - 12:00 Scribe: Christopher Bannon Tuesday, November 9, 2009 Proof: Brittany Paige Paugh Dr. Engler Tumor Viruses Page 3 of 5 i. This fact indicated that this is a single gene (many viruses around but no chickens infected at higher temperature) ii. Peter Vote = discovered deletion mutants of RSV 1. How long is the RNA in deletion mutants of RSV? a. Deletion mutant that is incapable of causing tumors is shorter than normal RSV. b. Missing segment = important for making tumors. iii. Winning the Nobel Prize = Harold Varmus and Michael Bishop (at UCSF) 1. Used reverse transcriptase (takes RNA and makes a DNA copy of it and its inserted in the chromosomes, remember its role in HIV) and make lots of copies (DNA) of normal RSV and then hybridize it to deletion RSV and all the segments that are the same sequence in both normal and deletion RSV, will hybridize and those that are different will not hybridize, but rather remain in solution = probe for what’s missing = what can make tumors) a. 1st experiment =went back and took probe from hybridization and bound it to regular RSV and call whatever it is an oncogenes (causes oncogenesis in chickens). 100% of probe binds to RSV RNA b. 2nd exp. Take chicken chromosomal DNA and discover 65% of probe binds to chicken DNA. What ever causes tumors in virus (whatever that gene is) is also present in chromosome of chickens/ cattle, mice, rats, and people i. Virus has taken something out of its normal chromosomal location and carried it in own genome and that now causes the virus to be able to transform cells (went to Sweden and go the Nobel prize for this) ii. Whatever the “probe” binds to in the genome is called a “proto-oncogene”, which is normally under control and doesn’t cause cancer. XV. [S13] Oncogene-encoding viruses a. Examples of RNA tumor virus i. RSV has 3 genes for the virus and the src gene 1. Src gene is the oncogene that causes sarcoma (solid tumors) in chickens 2. If infect chicken with src = oncogene forms tumors in host 3. Many times viruses are defective (taken out normal part of genome req. for growth and replace with oncogenes) This requires that you have mixtures of wild type viruses and these oncogenes carrying viruses to be able to infect cells in culture on in the wild. XVI. [S14] Chronology of understanding oncogenes a. We talked about proto-oncogenes already XVII. [S15] Genetic changes convert a Proto-oncogene into an Oncogene a. How does proto-oncogene become an oncogenes i. Normally for proton-oncogene to change into an oncogenes, a genetic change must occur ii. Epidermal Growth Factor receptors (EGF) normally recognize epidermal growth factor and its binding signals the cells to start growing. It has components on outside and inside cell, which transduce the signal. In the oncogene erbB, there has been a deletion that removes the epidermal growth factor sensing part of the receptor, and turns on the part inside the cell; and the protein thinks that the receptor is always bound, causing it to think that the EGF is always bound. So that’s one way to change to a proto-oncogene to change whatever it is that senses iii. Another way is to make a new receptor 1. Her 2 Neu = associated with breast cancer. 2. Neu oncogenes = another receptor on cell surface (single change from Val to Gln), although no deletion/ rearrangement, this single mutation always leaves the receptor “on” = inappropriate growth signal. XVIII. [S16] Proton-oncogenes mainly encode components of growth factor signal transduction pathway a. A lot of proteins involved in signal transduction from outside to inside the cell can be affected (are proto- oncogenes that can be turned into oncogenes if they become mutated) XIX. [S17] Four classes of oncogenes a. *****Know what each of 4 classes is and 1 example of how that oncogene works****** b. 1.) Things that pretend to be growth factors (ex. EGF receptors become oncogene when remove external portion of receptor protein) i. If virus starts making a protein which the receptor thinks is a GF, so that it now binds to the receptor, turning the receptor on, transducing a signal ii. These are rare but it is a phony growth factor sending an inappropriate single b/c its disconnected from normal cycles (acts like a platelet derived GF, it goes to the receptors and falsely represents PDGF) XX. [S18] Sis oncogenes acts like growth factor PDGF Fundamentals II: 11:00 - 12:00 Scribe: Christopher Bannon Tuesday, November 9, 2009 Proof: Brittany Paige Paugh Dr. Engler Tumor Viruses Page 4 of 5 a. – Takes the PGDF receptors and says its PGDF when in fact it comes from a virus XXI. [S19] Class Two: Mutated Receptors a. These are receptors that have lost their ability to sense the external environment of the cell and they remain “on” at all times b. – ErbB = deletion cell thinks there’ EGF present at all times causing the transducer protein to be “always on”. Can’t sense whether real ligand is there, and it thinks that it is “on” all the time XXII. [S20] Genetic Changes convert a Proto-oncogene into an Oncogene a. – Both of 2nd class of oncogenes = mutated receptors XXIII. [S21] Class Three: Intracellular transducers a. It’s called the transducer molecule and there are 4 kinds of transducer molecules. Once inside the cell these intracellular transducers are passing along the signals through cascade pathways to the nucleus i. Tyr kinase ii. Ser/Thr kinases iii. RAS proteins (remember Ras oncogene) iv. Phosopholiase C b. These are signal transducers which send the wrong signal, getting the message that cell should grow, at times when this signal should not be expressed by the cell. c. Don’t sense that it is not time to be on  inappropriate proliferation stimulus XXIV. [S22] Proto-oncogenes mainly encode components of growth factor signal transduction pathways a. If have mutation in these pathways, they can become oncogenes XXV. [S23] Class Four: transcription factor oncogenes a. Transcription factor AP1 has 2 potential oncogenes: Jun and Fos, if either has a mutation, the transcription factor works at inappropriate times. i. Myc is another famous oncogenes, involved in transcription ii. erbA = a receptor and a transcription factor b. XXVI. [S24] The same oncogenes can be found in more than one virus isolate a. You can find lots of different isolates of the same oncogene in different viruses (3 different isolates of erbB) all work in same way. Here’s a whole bunch of different one’s for Myc. b. Acute transforming viruses = carry oncogenes within them performing 1 of 4 tasks i. Acts like a phony growth factor ii. Acts as a mutated receptor that is always on. iii. Acts as signal transducer that is always on iv. Acts as signal transcription factor that sends inappropriate stimuli c. XXVII. [S25] Retroviruses can cause tumors in two ways: a. Non-acute transforming viruses do NOT carry any oncogenes but they can cause tumors but much more slowly than acute XXVIII. [S26] Oncogenesis by virus insertion a. –Transform through insertional mutagenesis = insert DNA copy of viral genome into host chromosome, interfering with regulation of near by genes b. They can cause chromosomal rearrangements or gene amplification c. Lots of ways that they can act, but again they do not carry oncogenes themselves but because of where they’ve been inserted they cause messed up gene regulation XXIX. [S27] How do tumors (and viruses) overproduce oncogenes proteins? XXX. [S28] Retrovirus life cycle requires integration into the chromosome XXXI. [S29] Insertional activation of proto-oncogenes a. Retroviruses have enhancers =genetic elements which bring transcription factors into the area b. Insertion of virus near a proto-oncogene, its enhancer can then turn on the nearby proto-oncogene at inappropriate time c. It can also insert its promoter so that transcription initiates from the viral promoter instead of the normal one in the host chromosome (promoter is on most of the time instead of being turned off until needed) d. It can mess up processing of RNA (piece gests transcribed along with proto-oncogene affecting splicing, its ability to be on, or be degraded) e. It could insert within an oncogene or within a tumor suppressor gene and then turn it off f. So that’s chronic viruses XXXII. [S30] Some DNA tumor viruses block tumor suppressor pathways a. Acute carry oncogenes; have 4 mechanisms (imitation of GF, Mutated Receptors always on, Transducer protein, or Mutated Transcription factor) Fundamentals II: 11:00 - 12:00 Scribe: Christopher Bannon Tuesday, November 9, 2009 Proof: Brittany Paige Paugh Dr. Engler Tumor Viruses Page 5 of 5 b. Chronic ones work indirectly by activating a proto-oncogene nearby i. Can lead to tumor if near proto-oncogenes ii. That’s the RNA tumor viruses now onto DNA tumor viruses XXXIII. [S32] Many DNA tumor viruses encode proteins that bind and sequester Rb a. DNA tumor viruses work by inactivating tumor suppressors, causing a “loss of function” b. These mutations are loss of function, in normal tumor must knock out both copies of tumor suppressor so can remove inhibition mechanism for cell regulation and control c. Lots of DNA tumor viruses i. Adenoviruses (came from tonsils and adenoids) 1. In the past there were no good antibiotics/treatments for tonsillitis; so they’d just yank em’ out. 2. When remove the tonsils, often times it was possible to culture adenovirus out of them ii. Gardasil protects against the high risk serotypes of HPV iii. Papova viruses (small DNA viruses that can infect people) 1. Simia Virus 490 = monkey virus 2. JKC and BK affect humans a. Each of these are small DNA viruses that can affect animals or people iv. Each of these viruses has a protein that can sequester Retinoblastoma protein (it and all its family members to inactivate Rb protein)). XXXIV. [S33] Retinoblastoma a. Each has proteins which can sequester retinoblastoma protein b. Rb = large tumor in the eye; rare ocular tumor that often develops in childhood i. 1/20,000 affected ii. Hereditary and nonhereditary forms (nonhereditary form is usually a single tumor) iii. Caused by a deletion in chromosome 13 (in both chromosomes) 1. Delete both copies of Rb protein which is a tumor suppressor 2. Does not cause Retinoblastoma alone, but it is also implicated in lung and breast cancer (known to be mutated) 3. Rb protein regulates cell replication 4. Viruses have figured out ways to inactivate the Rb protein to allow proliferation and growth. a. Rb wants to stop the cell from growing by inactivating Rb, the viruses can make it grow b/c they are DN viruses, using their own enzymes, they force the cell into S phase by controlling levels of DNA enzymes of the host cell. Eliminating these enzymes forces the cell through the cell cycle and allows production of more viral DNA iv. Goes back to slide 32 (c. v.) XXXV. [S34] Retinoblastoma gene (Rb) XXXVI. [S35] Rb shuts off cell proliferation by binding to E2F (a transcription factor) a. Function of Rb in a normal cell is to keep DNA synthesis turned off by binding transcription factor E2F (keeping it bound up) but at right time the Rb protein is phosphorylated, which makes it inactive allowing traveling into S phase and DNA synthesis eventually. And that’s where viruses want to be. Over in DNA synthesis b. Viruses able to inactivate Rb protein, not by phosphorylation, but rather by binding it up preventing it from adhering to E2F, making cell think it is about to go into S phase i. Preferential binding for Rb to viral protein ii. Inappropriate high levels of E2F promoting cell proliferation XXXVII. [S36] Viral proteins sequester tumor suppressors to promote cell proliferation XXXVIII. [S37] Myc acts as a transcription factor to activate cell proliferation genes XXXIX. [S38] How do oncogenes and tumor suppressors work? a. Oncogenic gain of function mutation telling the cell at wrong time, that its time to grow (through 1 of 4 mechanisms described earlier) they fake the cell out b. Tumor Suppressors, which act like “brakes”, DNA tumor viruses interfere with ability to keep cell turned off i. They sequester Rb protein, p53 protein, preventing it from doing its normal function as cell does not see any Rb or p53  shift into S phase 1. Cells grow out of control, because there is no Rb or p53 which would indicate that the cell should NOT move into S phase ii. Oncogenes are dominant (need the protein there to make things happen) iii. Tumor suppressors = both copies of gene need to be removed to “make the brakes come off” c. We know a lot of how cancer works b/c of DNA tumor viruses and RNA tumor viruses and our ability to manipulate them as genetic elements. XL. [S39] Summary Fundamentals II: 11:00 - 12:00 Scribe: Christopher Bannon Tuesday, November 9, 2009 Proof: Brittany Paige Paugh Dr. Engler Tumor Viruses Page 6 of 5 a. RNA Tumor Viruses, exert their affects on growth signaling. (GF’s, GF Receptors, Transducers, and Transcription Factors) Like adding gas to system = suck up glucose and AA to allow growth b. DNA tumor viruses = take brakes off. c. Very different mechanisms but viruses help us understand how cells can deviate from normal life cycle. d. **** Need to know 4 classes of oncogenes, e. **** Need to know how they work in broadest strokes and an example of each case.

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