Part III – Alice’s Lab Experiments
Alice was excited about the HDACi lab. What if her class discovered an HDACi that could be used to treat cancer? Maybe even Wiggles! Alice reviewed the lab results from her class.
Alice’s cell biology class had been testing potential HDAC inhibitors that were synthesized by the organic chemistry class last year. The organic students had completed the synthesis of two potential HDAC inhibitors and named them Xyzi and QB.
The first experiment they conducted was an inhibition assay. HDAC1 protein was mixed with buffer and HDAC substrate. Two additional samples also contained either 13 uM of Xyzi or QB. All three samples were incubated for 30 minutes. The reaction was then stopped, the amount of product was determined, and the relative HDAC1 activity was calculated by dividing the amount of product produced in the presence of Xyzi or QB by the amount produced without either chemical (analog). Figure 1 gives the relative activity of HDAC1 with Xyzi or QB.
Figure 1. The effect of potential HDACi on HDAC 1 activity. Relative HDAC activity is calculated by dividing the amount of product produced in the presence of the potential HDACi by the amount produced without the potential inhibitor. One hundred percent activity means the potential inhibitor did not inhibit HDAC 1.
Questions
1. Based on the results in Figure 1, is Xyzi or QB more effective at inhibiting HDAC 1? Explain your answer.
2. In a follow-up experiment, Alice’s class determined that Xyzi killed cancer cells at a lower concentration than QB. Based on the results in Figure 1, why is it not surprising that Xyzi kills cancer cells at lower concentrations than QB?
3. Alice’s class was to design an experiment to determine if Xyzi and QB cause changes in gene expression like known HDACi. They decided to examine the expression of p21.
a. Briefly explain the role of p21.
b. Design an experiment to determine if Xyzi and QB each activate the expression of p21 in U937 cells. Your design must specify treatments, controls (positive and negative), and methods for determining expression.
Special instructions for Question 3b:
i. Keep the experimental design to one page and include the following information:
α. Your hypothesis and prediction. (One sentence for each.)
β. Explanation of the methods. A flow chart is best.
γ. Assuming p21 expression is activated:
– Draw the expected results in a form consistent with the methods.
– Explain the results.