BIO360: The Molecular Biology of Cancer

Fall 2014

Take-Home Exam 3

The goal of this take-home exam is to assess how well you are able to read and evaluate scientific literature because this is a central skill that you will need in whatever you do after NCC. For this to work, it is simply essential that you are the only one answering these questions. Please read the following statements. If you agree to these provisions, please sign and date below and turn this in with your completed take-home exam. As always, please ask if something is unclear.

·  You may not receive help from another person. This is true for any student enrolled in this course or from any other person at North Central or elsewhere. The only exception is that you are encouraged to ask Dr. Johnston for any clarification that is needed.

·  You may not provide assistance to any other member of the class. If anyone approaches you for help with this exam, you are obligated to report that immediately to Dr. Johnston.

·  You may use any published material or on-line resource to help you answer the questions, so long as you do not plagiarize that source. At the end of your answers to the exam questions, please provide a brief, informal list of all sources that you used.

·  Seeking or providing help to another student on the take-home exam will result in your failing BIO360 and being reported to the Dean’s Office for further disciplinary action, including the possibility of permanent dismissal from the College.

By signing below, I certify that I have neither received nor provided any help from another person in completing this assignment. I have read, understood and agree to abide by all of the above terms and conditions.

Printed Name: ______

Signed: ______Date:______


Read the following paper and answer the questions below.

Meng, X.W., B.D. Koh, J.-S. Zhang, K.S. Flatten, P.A. Schneider, D.D. Billadeau, A.D. Hess, B.D. Smith, J.E. Karp and S.H. Kaufmann. 2014. Poly(ADP-ribose) polymerase inhibitors sensitize cancer cells to death receptor-mediated apoptosis by enhancing death receptor expression. J. Biol. Chem. 289:20543-20558.

Try not to analyze what the authors of the paper say in the text – rather analyze what the data say. Please type your answers. You are welcome to augment your written answers with hand-drawings if that helps explain your ideas. Don’t quote this paper or any other. Be sure to refer to specific data contained in this paper frequently to support your answers. For example, don’t say “the data are in Figure 12” but rather say “the data are in the first four lanes of Figure 12C, comparing the western blot on the top versus the western blot on the bottom”. Please put your name only on the signature page only and not on the paper itself. All answers are due at noon on Wednesday November 5. Email submissions are fine, but still must meet the same deadline.

1. Figure 1A was generated using a FACS machine but they didn’t use propidium iodide as a stain, as we have seen in other papers. What molecule is being detected on these cells? How does this stain connect to their conclusion about apoptosis? (5 points)

2. Figure 4A seems to show that olaparib treatment does not alter the levels of FADD. Yet the graph on the right side of Figure 5A seems to show that olaparib treatment roughly doubles the levels of FADD! Please either explain how both of these conclusions can be true or explain how Meng et al. made an error in their analysis. (5 points)

3. Figure 7A reports on the levels of Fas and DR5 mRNA. From a single mRNA sample, what was different in the experiments to specifically detect either the Fas mRNA or DR5 mRNA? Why is there no error bar on the DMSO-treated samples? Moving forward from this observation, what hypothesis are Meng et al. exploring in the experiments shown in Figures 7B and C? These cells were treated with two compounds (other than the DMSO or olaparib): actinomycin D and Q-VD-OPh. Please briefly explain why each of these compounds was added to this experiment. (10 points)

4. Identify the figure (or part of a figure) that supports each of the following five conclusions from this paper. In each case, provide a few sentences to explain how the data lead to this conclusion. (10 points)

A.  PARP inhibition by olaparib does not lead to a change in the overall quantity of the transcription factor Sp1 in these cells, but rather it just changes it’s subcellular localization.

B.  Changes to the subcellular localization of Sp1 leads to more transcription of the TNFRSF10B gene, encoding DR5.

C.  As a result, more DR5 is found in high concentrations on the surface of more of these cells.

D.  With more DR5 on the cell surface, these cells are more sensitive to TRAIL-induced apoptosis.

E.  Simultaneously inhibiting PARP while activating DR5 decreases the ability of three different acute myelogenous leukemia cells to grow in a colony-formation assay.

5. Consider Figure 5C. What instrument was used to generate these data? The x-axis obviously shows fluorescence levels but what is the source of the fluorescence in this experiment? Why did the authors use a ten-fold higher concentration of veliparib than olaparib in this experiment? (5 points)

6. I find the data in Figure 9C to be less-than-impressive. A darker exposure of the gel would help, but some additional controls would also be useful. Describe two additional controls that they could have run with this ChIP assay. Be sure to explain how each would improve their overall conclusion. (5 points)

7. What else do you want to know? Good papers always tell us something new about the world which has the effect of raising new questions. Begin by putting forth a specific hypothesis or question. In your answer, list your hypothesis or question in one sentence and underline that sentence. Design an additional experiment or two that will significantly extend the results of Meng et al. Be original and don’t just re-state your answer to an earlier question on this exam.

Be sure to explain the rationale behind your hypothesis, briefly outline an experiment or two and show why the new results will be important. Be very clear about your dependent and independent variables. In other words, what will you alter and how you will alter it? What will you measure and how will you measure it? In your answer, cite at least one primary research paper other than those that we’ve looked at as a class. (10 points)