Answer Key: Regulation of Cell Division

BIOL230W Week 9 – Regulation of Cell Division Mechanically, the cell cycle results in equal separation of DNA between two new daughter cells. To ensure that this process occurs correctly, thereby preventing cancer, cells have several regulatory steps. This week we will explore the molecules and steps of cell cycle regulation. Day 1 Learning Objectives 1. Describe the timing of the three major checkpoints in relation to the cell cycle and what they are monitoring. 2. Compare and contrast cyclins and cyclin-dependent kinases. 3. Define mitogen (example myc) and entry into the cell cycle. 4. Compare and contrast proto-oncogene, oncogene, and tumor suppressor genes. Recall Activity: Recap of Cell Division 1. Briefly describe the result of each of the stages of the cell cycle: A. G1: growing and preparing for S-phase. B. S: replication of DNA and formation of sister chromatids C. G2: growing and preparing for M-phase. D. Mitosis: separation of sister chromatids into 2 new daughter cells 2. Thinking about the Week 9 Exploration Homework, what types of problems are cells monitoring at each checkpoint between these stages? A. G1àS checkpoint: nutrients and environmental cues to make s-phase appropriate. B. G2àMitosis checkpoint: successful replication C. Metaphase checkpoint: kinetochores are all attached to microtubules. Mini-Lecture: Intro to Cyclin-dependent Kinases (CDKs) and Cyclins CDKs and cyclin work together to regulate the cell cycle. The diagram below shows the levels of one CDK and its related cyclin. The shaded area indicates maximum activity of the CDK. 3. How does the concentration of the cyclin relate to the concentration of the CDK throughout the cell cycle? CDK concentration is constant. 4. At which stage is the CDK most active? BIOL230W Week 9 – Regulation of Cell Division M phase 5. At which stage is the concentration of the cyclin the highest? M phase 6. Summarize how CDKs and cyclins function to regulate the cell cycle? Cyclin and CDK will bind together to form an activated complex that promotes cell cycle, push cycle into and through M-phase. 7. How are CDKs inactivated? When cyclins are degraded Small Group Activity: Myc Signaling Pathway (Entering the Cell Cycle) Epidermal growth factor (EGF) is a protein mitogen. It is expressed and released by cells via exocytosis and causes increased cell division in target cells. Epidermal growth factor receptors (EGFR) are related to receptor tyrosine kinases. Several kinases are involved in this pathway and ultimately result in phosphorylation of Myc to an active state. Activated Myc can bind to DNA in enhancer regions and upregulate gene expression of proteins required for the cell cycle. 8. What is the ligand of the pathway? Why is it a mitogen? Epidermal Growth Factor – it causes increased cell division (mitosis) 9. Which type of signaling molecule is Myc: ligand, receptor, second messenger, effector? Activator (effector) – increases gene expression. 10. What would be the result of preventing GDP:GTP exchange for the protein RAS? RAS protein would not be activated, and no further reaction would occur. 11. What would be the result if Myc was mutated so that it is always in the activated conformation? It would always be promoting the cell cycle even if the pathway is off – causes unregulated cell division/cancer. BIOL230W Week 9 – Regulation of Cell Division Mini-Lecture: Mitogens 12. Describe the difference between proto-oncogene and oncogene. Proto-oncogenes support normal cell division – oncogene is a mutated version that does not correctly regulate cell division. Day 2 Learning Objectives 1. Describe the role of p53 in the cell cycle. 2. Compare and contrast loss-of-function and gain-of-function mutations and their effect on gene expression. 3. Explain how the balance of kinase and p-proteins affects the cell cycle. Small group activity: The Role of P53 in the Cell Cycle Caption: Alleles of the p53 gene were selectively disrupted in a line of human cells and then monitored after exposure to DNA-damaging gamma (γ) radiation to determine what proportion of the cells entered mitosis (mitotic index). The shaded squares represent cells with two normal alleles of the p53 gene. The half-shaded squares represent cells with one normal and one loss-offunction mutated allele of the p53 gene (note that some of the half- shaded squares are covered by the shaded squares – at 60 hours you can see an example of a half-shaded square). The unshaded squares represent cells in which both alleles of the p53 gene have loss-of-function mutations. 13. In this work, there are three different cell lines that have been experimentally treated. What is the treatment? Exposure to gamma radiation that damages DNA 14. Which cell line(s) (represented by shaded, half shaded or unshaded boxes) express a functioning p53protein? Shaded and half-shaded, only need one functioning allele for p53 to work. 15. Which cell line(s) (represented by shaded, half shaded or unshaded boxes) represents wildtype function of p53? What is the wildtype cell behavior after exposure to radiation? Shaded and half shaded. BIOL230W Week 9 – Regulation of Cell Division 16. How many wildtype p53 alleles do cells need to function properly? Use evidence from the figure to support your claim. One, because mitotic index was the same for normal (non-exposed) and loss of function of ½ alleles. 17. Based on the figure, what role does the p53 protein play in cell division? Why might a cell need to stop dividing? P53 blocks the cell cycle to prevent unregulated cell division (prevents development of cells with damaged DNA) Mini-Lecture: p53 and P-Proteins 18. What is the main function of p53 in the cell? Blocks the cell cycle to prevent development of cells with damaged DNA. 19. How does expression of p21 inhibit the cell cycle? P21 is an inhibitor (expression increased by p53), so it inhibits CDKs. Small group activity: Loss-of-function and Gain-of-function of mutations 20. List the wildtype function of Myc and p53. Then hypothesize the results if Myc or p53 acquired a mutation that inhibits function (i.e., loss -of-function mutation). Myc: promotes cell cycle – mutation will cause too little cell division. p53: Blocks the cell cycle via upregulation of p21 (inhibitor that inhibits CDK) – if function is lost there will be unregulated cell division/cancer. 21. Remember that each gene has two alleles (hint – a protein can be produced from each allele). How many alleles must have a loss-of-function mutation to result in the hypothesized function from Q20? 2 alleles 22. Using the example above, which mutation (Myc or p53) will lead to cancer? P53 – unregulated cell division 23. Hypothesize the results if Myc or p53 acquired a mutation that created new function (i.e., gain-offunction mutation that causes it to do more of what it already does).