1. Describe in detail the relationships between the DNA damage response and genomic instability—distinguish between numerical, structural and mutational instability. Using sources and examples outside of those discussed in class, illustrate how the DNA damage response proteins contribute to carcinogenesis. How does this support CIN as an “enabling characteristic” of cancer cells? (10 points)
2. How would you propose to exploit genomic instability in cancer therapy? (6 points). Again—go beyond the notes.
3. Why are mutant tumor suppressor genes transmitted through the germline while mutant proto-oncogenes usually are not? (3 points)
4. The GTPase Ras is mutated in approximately 30% of all human cancers.
A) What is the most common mutation that converts the RAS proto-oncogene into an oncogene and why does this mutation render Ras oncogenic? (2 points)
B) Attempts to develop drugs that directly target mutant Ras have, for the most part, been unsuccessful. Why is this the case and what is one alternative strategy for inhibiting signaling pathways driven by oncogenic Ras? (3 points)
5. A point mutation within the coding sequence of an enzyme that confers gain-of-function is one well established mechanism for oncogene activation. Describe two other mechanisms of oncogene activation. For each mechanism, give a specific example that has been reported in cancer. (4 points)
6. If a mutant tumor suppressor gene (TSG) undergoes loss-of-heterozygosity in one out of 104 or 105 cells, and if an adult human is comprised of more than 1013 cells, why doesn’t a person who inherits a single mutant TSG develop thousands of tumors? (3 points)
7. Wilms tumor (pediatric nephroblastoma) can be caused by mutation of the WTX tumor suppressor gene. However, in contrast to classical tumor suppressor genes that require two “hits” for inactivation, WTX can be inactivated by a monoallelic “single-hit” event. Why is this the case for this particular TSG? (3 points)
8. You have identified a gene in the lab that you hypothesize is a novel tumor suppressor gene. Briefly outline a cell-based experiment to test your hypothesis. In your answer, define the criteria that need to be satisfied before you would be confident in classifying this gene as a TSG. (6 points)
Frank Purdue recently observed an increased morbidity and mortality rate at various chicken farms. Blood samples from sick chickens contained extremely elevated levels of abnormal lymphocytes, and these chickens eventually developed an incurable leukemia. 10 un-affected chickens were injected with serum derived from a leukemic chicken. Initially, all 10 chickens remained unaffected; however, by four months, 7 of these chickens began to develop leukemia. Electron microscopy of serum samples derived from leukemic chickens showed the presence of particles that were approximately 100nm in diameter. Incubation of cultures of primary chicken lymphocytes with serum from affected chickens resulted in the de novo synthesis of similar particles that were secreted from infected cells. Similar results were seen when cells were treated with serum that had first been filtered to remove all bacteria and fungi. Long term cultures of the infected, cultured lymphocytes continued to produce particles in the absence of obvious effects on cellular physiology. When particle-containing supernatants were treated with ether or chloroform and then added to cultured chicken lymphocytes, no newly synthesized particles were produced. Likewise, no particles were produced when these supernatants were added to cells that were subsequently treated with a combination of high levels of BUdr (bromodeoxyuridine) and ultraviolet light (cells were not killed by this treatment). Proteinase K treatment of concentrated supernatants revealed that the infectious particle contained a nucleotide genome; this genome was RNAse A sensitive. (NOTE: YOUR ANSWERS TO THE FOLLOWING 4 QUESTIONS CANNOT EXCEED TWO PAGES (IN TOTAL, NOT PER QUESTION); 0.5 INCH MARGINS, 12 POINT, SINGLE SPACE.
(1). The characteristics of the infectious agent described above support the hypothesis that it is a virus. What viral family does it likely belong to, and why? (Include in your answer the significance of “filterable agent”, sensitivity to chloroform, ether and BUdr/UV and the type of genome that is encapsidated—a general virology textbook and Howard Temin can help you answer this question). (20% of total points)
(2). Is it likely that this virus encodes a strong oncogene? How did you reach this conclusion? (15% of total points)
Using standard hybridization techniques, you demonstrate that the genome of your virus hybridizes with lymphocyte DNA extracted from chickens that have leukemia caused by this virus. However, the genome does not hybridize with non-lymphocyte cells derived from the leukemic chickens or with any cells, including lymphocytes, derived from uninfected chicken DNA.
(3). Why does the viral genome hybridize with lymphocyte DNA from the infected chickens and not with DNA from any other chicken cells (from either infected or uninfected chickens)? How do these hybridization studies support your answers to questions 1 and 2? (30% of total points)
(4). Provide a plausible hypothesis for how this infectious agent could cause leukemia. (Note that there is not necessarily an absolutely correct answer. One reasonable hypothesis is acceptable.) (35% of total points)
10. Discuss significance of cell cycle checkpoint studies to cancer therapy.
(Answer the following questions in a 1/2 - 1 page essay.)
(1). Describe similarities and differences among mismatch repair, base excision repair and nucleotide excision repair (3 points)
(2). Explain why deficiencies in transcription-coupled DNA repair leads to UV-sensitivity, but not to
cancer (3 points).
(3). Design experiments to test the involvement of a given protein in DNA repair, genomic stability and
tumorigenesis (4 points)
(4). Describe the role of oxidative DNA damage in predisposition to human cancer (4 points)
(5). Describe all experimentally proven connections of the system of homologous recombination and also non-homologous end-joining with tumorigenesis. Propose hypothetical connections, which don’t have experimental support but are consistent with known properties of these systems (Essay-10 points).
You have just performed a microarray analysis comparing genes expressed in normal tissue with tumor samples and got a list of genes regulating apoptotic pathways that are altered in the tumor tissue.
Refer to the list below:
Tumor 1 genes upregulated:
Tumor 2 genes downregulated:
a. Explain the function of each gene in regulating apoptotic pathways (6 points).
1. Mechanism of action (including move cell closer to or farther from death threshold)
2. Does protein belong to intrinsic or extrinsic pathway?
b. Which of these tumors (Tumor 1 vs Tumor 2) would you expect to be more resistant to apoptosis induced by ionizing irradiation? Why? (1 points)
c. Your microarray picked up an unknown gene "DRGN1". Design an experiment to test whether this gene regulates apoptosis (be sure to include how you would measure apoptosis) (1 points)
d. Your results suggest that DRGN1 is a regulator of apoptosis. Design an experiment to test whether it regulates the intrinsic AND extrinsic death pathways. (2 points)
BH3 mimetics are thought to be potentially a breakthrough in anticancer therapy. Explain why (especially in the context of p53 status). (2 points)
(Can use diagrams to support written answer)