Cell Biology Final Assignment Background More than one in ten women develop breast cancer during life, but death rates have declined steadily because of earlier detection and new treatments. As with other forms of cancer, breast cancer results from mutations in DNA. When enough mutations accumulate, a cell can be more readily triggered to reproduce by division and ignore signals that trigger death by apoptosis. This rapidly dividing cell forms a tumor, which differs in structure and function from normal tissue. Ultimately, some of these cells leave the tumor and enter the bloodstream. Death results when these cells circulate to vital organs and interfere with their functions. Scientists have identified a signaling pathway called the GPR/TRPV signaling pathway that regulates reproduction and death of cells in breast tissue (Figure 1). In the absence of the signaling molecule S1 (Figure 1, left panel), the receptor GPR is in its inactive form. The subsequent structures: G-complex, Erk1, and p38, are also in their inactive form, thus cell division does not occur. Similarly, TRPV is in its inactive form in the absence of S1. Consequently, the concentration of calcium ions (Ca2+) does not increase and the structures ATM and p53 are in their inactive form, thus cell death does not occur. When S1 is present (Figure 1, right panel), the following steps occur: GPR signaling pathway 1) A signal called S1 binds to the receptor GPR, inducing a conformational change. 2) The active form of GPR binds and phosphorylates the G-complex. 3) The active form of the G-complex binds and phosphorylates a kinase called Erk1. 4) The activation of Erk1 leads to the activation of a kinase called p38 by phosphorylation. 5) The activation of p38 leads to an increase in cell division. TRPV signaling pathway 1) A signal called S1 binds to the receptor TRPV, preventing a conformational change. 2) The inactive form of TRPV does NOT open a channel to allow Ca2+ to move into the cell. 3) Since Ca2+ levels in the cell do NOT increase, a kinase called ATM can NOT become activated by phosphorylation. 4) Since ATM is NOT activated, this does NOT lead to the activation of p53 by phosphorylation. 5) Since p53 is NOT activated, this does not lead to an increase in cell death by apoptosis. Figure 1. The GPR/TRPV signaling pathway that regulates cell division and cell death of cells found in breast tissue. Left: When no signaling molecule (S1) is present, all proteins and processes in the signaling pathway are inactive and [Ca2+] in the cell do not increase. Right: When the signaling molecule S1 is present, it binds to the receptor GPR, inducing a conformational change. The activation of GPR results in the phosphorylation of the proteins/structures downstream of GPR – specifically the G-complex, Erk1, and p38, ultimately resulting in an increase in cell division. Additionally, when the signaling molecule S1 is present, it binds to the receptor TRPV, preventing a conformational change. The inactivation of TRPV means that Ca2+ does not flow into the cell and thus the [Ca2+] inside the cell does not increase. Consequently, ATM and p53 are not phosphorylated, thus no cell death by apoptosis occurs. Designing an effective treatment for breast cancer requires an understanding of the signaling pathways that determine whether cells reproduce or die, such as the GPR/TRPV signaling pathway (Figure 1). Researchers discover effective treatments through experiments, testing each molecule to determine to what extent that molecule disrupts the function of signaling pathways responsible for inducing cancer as well as the mechanisms by which a given treatment works. Scenario You work for a drug company that aims to develop a new treatment for breast cancer. Previously, scientists discovered that people with breast cancer have unusually high concentrations of endocannabinoids. Researchers at your company hypothesized that endocannabinoids interfere with the GPR/TRPV signaling pathway that controls cell division and cell death in the cells of breast tissue. However, the exact mechanisms remain unclear. If you can determine the mechanism by which endocannabinoids stimulate cell division and inhibit cell death, you can identify potential treatments that would treat breast cancer. To complete this assignment, you should follow the sequence of steps below: Step 1: Determine if receptor(s) in the GPR/TRPV signaling pathway bind to endocannabinoids. Step 2: Determine how the presence of endocannabinoids affects the activity of downstream proteins in the cytoplasm of the GPR/TRPV signaling pathway. Step 3: Evaluate potential treatments for breast cancer. Step 1: Determine if receptor(s) in the GPR/TRPV signaling pathway bind to endocannabinoids. Before we can start exploring potential cancer treatments, we will explore how (if at all) the presence of endocannabinoids affects the GPR/TRPV signaling pathway. We’ll start by determining if the receptor(s) in the GPR/TRPV signaling pathway bind to endocannabinoids and, if so, the impact of this binding on the activity of the receptors GPR and TRPV. The following experiment was conducted. Ten genetically identical cells were collected from healthy breast tissue. The relative activity (% of observed max) of the receptors GPR and TRPV were separately measured in these healthy cells. Then, the cells were exposed to an endocannabinoid suspected to be associated with causing breast cancer (called ENDO1) and the measurements were repeated. Using this information, answer the following questions. Part 1 – Effect of endocannabinoids on the activity of GPR Directions: For questions 1-5, download the Excel file, “Data: Effect of ENDO1 on Activity of GPR and TRPV,” in Canvas containing the activity (%) of GPR in the absence or presence of endocannabinoids (sample size = 10 activities per category, absence or presence of ENDO1). Use Excel for calculations, modeling, and graphing. Round all calculated values to the nearest tenth of a decimal place. For example, if you calculate the value as 5.252%, you would round your answer to 5.3%. 1. Create a plot of a linear relationship between the categorical independent variable (absence or presence of ENDO1) and the activity of GPR. Your plot should follow the formatting guidelines listed below. Formatting Instructions General ● Chart type: Line ● Quick layout: Layout 1 ● Y-axes title: “Mean relative activity of GPR (%)”; Font size = 32 ● Y-axis numbers: Font size = 24 ● X-axis title: “Absence or presence of ENDO1”; Font size 32 ● X-axis numbers: Font size = 24 ● Line: No line Marker ● Marker options: Built in, Filled in circle, Size = 10 ● Fill: Solid fill, Color = Black ● Border: Solid line, Color = Black Error bars → Standard deviation ● Direction: Both ● End style: Cap ● Error amount: Custom → Select the appropriate cell in Excel. ● Line: Solid line ● Color: Black ● Width: 2 pt Y-axis ● Bounds: minimum at 0, maximum at 100 ● Tick marks: Major = 10, Minor = 5 2. Based on the figure that you made in Question 1 describe how the absence or presence of ENDO1 affects the activity of GPR. In other words, how does the activity of GPR in the absence of ENDO1 compare to the activity of GPR in the presence of ENDO1? 3. Estimate the standard deviation of the linear relationship between the categorical independent variable (absence or presence of ENDO1) and the activity of GPR. Standard deviation = 4. Estimate the mean activity of GPR in the absence of ENDO1. Mean activity = 5. Estimate the mean activity of GPR in the presence of ENDO1. Mean activity = Part 2 – Effect of ENDO1 on the activity of TRPV Directions: For questions 6-10, download the Excel file, “Data: Effect of ENDO1 on Activity of GPR and TRPV,” in Canvas containing the activity (%) of TRPV in the absence or presence of endocannabinoids (sample size = 10 activities per category, absence or presence of ENDO1). Use Excel for calculations, modeling, and graphing. Round all calculated values to the nearest tenth of a decimal place. For example, if you calculate the value as 5.252%, you would round your answer to 5.3%. 6. Create a plot of a linear relationship between the categorical independent variable (absence or presence of ENDO1) and the activity of TRPV. Your plot should follow the formatting guidelines listed below. Formatting Instructions General ● Chart type: Line ● Quick layout: Layout 1 ● Y-axes title: “Mean relative activity of TRPV (%)”; Font size = 32 ● Y-axis numbers: Font size = 24 ● X-axis title: “Absence or presence of ENDO1”; Font size 32 ● X-axis numbers: Font size = 24 ● Line: No line Marker ● Marker options: Built in, Filled in circle, Size = 10 ● Fill: Solid fill, Color = Black ● Border: Solid line, Color = Black Error bars → Standard deviation ● Direction: Both ● End style: Cap ● Error amount: Custom → Select the appropriate cell in Excel. ● Line: Solid line ● Color: Black ● Width: 2 pt Y-axis ● Bounds: minimum at 0, maximum at 2 ● Tick marks: Major = 0.5, Minor = 0.1 7. Based on the figure that you made in Question 6 describe how the absence or presence of ENDO1 affects the activity of TRPV. In other words, how does the activity of TRPV in the absence of ENDO1 compare to the activity of TRPV in the presence of ENDO1? 8. Estimate the standard deviation of the linear relationship between the categorical independent variable (absence or presence of ENDO1) and the activity of TRPV. Standard deviation = 9. Estimate the mean activity of TRPV in the absence of ENDO1. Mean activity = 10. Estimate the mean activity of TRPV in the presence of ENDO1. Mean activity = Step 2: Determine how the presence of endocannabinoids affects the activity of downstream proteins in the cytoplasm of the GPR/TRPV signaling pathway. Now that you’ve determined if ENDO1 binds to either of the receptors in the GPR/TRPV signaling pathway, it’s time to determine if or how the presence of endocannabinoids affects the activity of downstream proteins in the cytoplasm of the GPR/TRPV signaling pathway. By answering this question, we will have a better understanding of how endocannabinoids such as ENDO1 affect the regulation of cell division and cell death in cells found in breast tissue. Researchers at the drug company followed up on their experiment (outlined at the beginning of Step 12). In this follow-up experiment, they measured the relative activity (% of observed max) of the proteins in the cytoplasm downstream of GPR and TRPV receptors in healthy cells in the absence and presence of ENDO1. They also measured the concentration of Ca2+ inside the cells (called intracellular Ca2+ concentration, [Ca2+i]. Their results are summarized in Figure 2 and Figure 3 below. Figure 2. The relative activity of three types of proteins/complexes in the absence or presence of ENDO1. The plots depict activities of G-complex, Erk1, and p38 (left to right). Activity scales from 0% to 100%. To make the data easier to see, a break in the y-axis omits the range of values between 25% and 70%. Figure 3. The intracellular concentration of Ca2+ ([Ca2+i]) and the relative activity of two types of proteins in the absence or presence of ENDO1. The plots depict activities of [Ca 2+i], ATM, and p53 (left to right). Activity scales from 0% to 100%. To make the data easier to see, a break in the y-axis omits the range of values between 25% and 60% (left plot) or between 25% and 70% (middle and right plots). Figure 4 (below) shows two signaling pathways. The left box shows a complete diagram of the signaling pathway in healthy cells (healthy signaling pathway). The middle box shows an incomplete diagram of the signaling pathway in tumor cells (cancerous signaling pathway). The right box contains a key to the symbols used to indicate the activation, inactivation, or phosphorylation of a protein/process in the signaling pathway. This figure is also provided as a PowerPoint file so you can copy, drag, and drop the symbols to complete the pathway. Figure 4. Signaling pathways in a healthy or cancerous cell found in breast tissue. Left: In healthy cells, the signaling molecule called S1 binds to the receptor GPR, initiating a series of steps that results in cell division occurring at typical rates observed in healthy cells. However, S1 also binds to and inhibits TRPV, preventing programmed cell death from occurring. Middle: In cancerous cells, the signaling molecule called ENDO1 binds either GPR, TRPV, both GPR and TRPV, or neither receptor. Ultimately, when ENDO1 is present, cell division occurs at excessive rates and programmed cell death does not occur, emblematic of what is observed in tumor cells. Right: A key to the symbols used to indicate the activation, inactivation, or phosphorylation of a protein/process in the signaling pathway. Previous research has documented the interactions between proteins/processes in the healthy signaling pathway. The proteins/processes in the left and middle boxes in Figure 4 are presented in the sequence in which they interact in this pathway. However, the middle box in Figure 4 omits information about the nature of the interactions between these proteins when ENDO1 is present. You must insert symbols in the middle box to indicate the interactions between proteins/processes in the presence of ENDO1. Using the results of your analyses in Step 1 and the data in Figure 2 and Figure 3, answer the following questions. Directions: For question 11, download the PowerPoint file, “Incomplete cancerous signaling pathway,” containing the incomplete diagram of the cancerous signaling pathway. Follow the steps described below to complete the pathway when ENDO1 is present. To add a symbol to the incomplete diagram, right-click on the symbol in the key and select “copy.” Then, paste this icon and drag it to the desired position in the incomplete diagram. Use the results of your analyses in Step 1 and the data in Figure 2 and Figure 3 to complete the cancerous signaling pathway. Keep in mind the following assumptions as you complete your diagram: (1) the proteins/processes in the signaling pathway are presented in the sequence in which they interact and (2) any receptor (GPR or TRPV) activated in the presence of ENDO1 can directly activate (or inhibit) only one type of protein or process in the cytoplasm. 11. Upload your completed cancerous signaling pathway in the presence of ENDO1. Use the results of your analyses in Step 1 and the data in Figure 2 and Figure 3 to help you complete the signaling pathway. 12. Explain how the results of your analyses in Step 1 support the diagram you uploaded in Question 11. 13. Explain why each plot in Figure 2 supports the diagram you uploaded in Question 11. 14. Explain why each plot in Figure 3 supports the diagram you uploaded in Question 11. Step 3: Evaluate potential treatments for breast cancer. Now that you have determined how an endocannabinoid such as ENDO1 can induce breast cancer via the signaling pathway in Figure 4, we’re ready to evaluate potential treatments for breast cancer that occurs via disruptions in the signaling pathway in Figure 4. The drug company you work for has asked you to evaluate two related cannabinoid molecules that could help treat breast cancer. Since 1975, researchers have been aware that cannabinoids can significantly inhibit tumor growth. More recently it was found that phytocannabinoids (cannabinoids produced by plants) are toxic to cancer cells because they interact with the human endocannabinoid system, upregulating immune responses that protect the body from the development/progression of cancer cells. Your company has identified two phytocannabinoids, CBD1 and CBD2, that they would like you to evaluate as potential treatment for breast cancer. The following experiment was conducted to evaluate these molecules. Ten genetically identical cells were collected from cancerous breast tissue. The relative activity (% of observed max) of cell division and cell death were measured in these cancerous cells while ENDO1 was present. Then, the cells were separately exposed to two phytocannabinoids, CBD1 and CBD2, and the measurements were repeated after each exposure. The results of these experiments are shown below. Figure 5. The relative activity of cell division and cell death in the absence or presence of CBD1. The plots depict activities of cell division and cell death (left to right). Activity scales from 0% to 100%. To make the data easier to see, a break in the y-axis omits the range of values between 25% and 70%. Figure 6. The relative activity of cell division and cell death in the absence or presence of CBD2. The plots depict activities of cell division and cell death (left to right). Activity scales from 0% to 100%. To make the data easier to see, a break in the y-axis omits the range of values between 25% and 70 (left plot) and 25% and 60% (right plot). Using the data in Figure 5 and Figure 6, evaluate the two potential treatments for breast cancer and recommend which one would be a better treatment. Be sure to provide your reasoning, highlighting the relevant evidence supporting your claim. We recommend that you reread the background at the beginning of this document before you answer the following questions. 15. Construct a claim that answers the question “Which molecule, CBD1 or CBD2, is a better treatment for breast cancer caused by disruptions to the signaling pathway in Figure 4 by ENDO1?” 16. What evidence in Figure 5 supports your claim in Question 15? 17. What evidence in Figure 6 supports your claim in Question 15? 18. Put it all together: Why, from a biological perspective, did you recommend one molecule over the other in Question 15? Your answer should minimally explain (1) what the ideal treatment would look like and (2) connect the evidence in Figure 5 and Figure 6 to your claim in Question 15. CANCEROUS SIGNALING PATHWAY ICONS FOR COMPLETING THE CANCEROUS PATHWAY ENDO1 Icon indicates activation of a protein or process Plasma Membrane GPR Icon indicates inhibition of a protein or process TRPV G-complex ↑Ca2+ Erk1 ATM p38 p53 Excessive Cell Division No Apoptosis (Cell Death) P Indicates phosphorylation of a protein Presence or Absence Mean Relative of ENDO1 Activity (% of observed max) of GPR Absence 6.6 Absence 7.4 Absence 8.6 Absence 6.3 Absence 8.9 Absence 9.4 Absence 9 Absence 6.7 Absence 4.5 Absence 5.6 Presence 98.9 Presence 99.8 Presence 99.5 Presence 99.3 Presence 98 Presence 97.5 Presence 98.2 Presence 98.1 Presence 97.8 Presence 99.5 Mean Relative Activity (% of observed max) of TRPV 1.6 1.3 1.1 1.7 1.5 1.6 1.4 2 1.3 1 1.9 0.6 1.8 0.9 1.4 1.7 1.5 1.3 1.4 1.3
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