Lecture+2_The+Cell+Cycle.pptx

Optional accompanying reading Pharmacology 4001: Mechanisms of Drug Action Chapter 17 Lecture 2: The Cell Cycle January 18, 2024 What is Pharmacology? “The study of the effect of chemical substances on the function of living systems.” –Pharmacology, 5th edition, Range et al., 2003 What is Cancer? Ca...

Optional accompanying reading Pharmacology 4001: Mechanisms of Drug Action Chapter 17 Lecture 2: The Cell Cycle January 18, 2024 What is Pharmacology? “The study of the effect of chemical substances on the function of living systems.” –Pharmacology, 5th edition, Range et al., 2003 What is Cancer? Cancer cells are defined by two heritable properties: 1. They reproduce in defiance of the normal restraints on cell growth and division 2. They invade and colonize territories normally reserved for other cells What is Cancer? Cancer cells are defined by two heritable properties: 1. They reproduce in defiance of the normal restraints on cell growth and division How? To understand this disease state, we need to understand how healthy cells grow and divide. Cell growth and division occurs in stages called the ‘cell cycle.’ All anti-cancer drugs directly or indirectly slow progression through the cell cycle. So, let’s talk about how it works! 2. They invade and colonize territories normally reserved for other cells The Cell Cycle Optional accompanying reading: Chapter 17 Figure 17.4 of B Alberts, A Johnson, J Lewis, M Raff, K Roberts and P Walter “Molecular Biology of the Cell” 6th edition 2014, Garland Science The Cell Cycle Accompanying lecture notes now available on Canvas Preview The major regulatory phase in mammalian cells is at the G1-S boundary The machinery that controls this transition are cyclin-dependent kinases How are cyclin-dependent kinases related? 1. Cyclins 2. Activating phosphates 3. Inhibitory phosphates 4. Phosphatases that remove inhibitory phosphates 5. Cyclin-dependent kinase inhibitors (CKIs) CKI transcription (initiated by transcription factor p53) CKI proteolysis (initiated by a ubiquitin ligase) Learning Objectives 1. Describe how the cell cycle is regulated. 2. List what needs to ‘go wrong’ for a cancer to develop. 3. Identify types of proteins could potentially be targeted in new cancer therapies. The Cell Cycle Figure 17.4 of B Alberts, A Johnson, J Lewis, M Raff, K Roberts and P Walter “Molecular Biology of the Cell” 6th edition 2014, Garland Science Visualizing The Cell Cyle in an Onion Root Tip Not exciting to look at under a light microscope in high school biology but the phase most relevant to cancer therapy! Figure 17.4 of B Alberts, A Johnson, J Lewis, M Raff, K Roberts and P Walter “Molecular Biology of the Cell” 6th edition 2002, Garland Science The Cell Cycle Growth phase after DNA replication DNA gets synthesized S = synthesis Figure 17.4 of B Alberts, A Johnson, J Lewis, M Raff, K Roberts and P Walter “Molecular Biology of the Cell” 6th edition 2014, Garland Science No ‘typical’ cell cycle time in humans. Cells divide at different rates. Fast growing human cells divide in ~ 24 hours. Of those hours, ~1 hr in M phase ~12 hr in S Phase and the rest spent in growth phases. Growth phase after a cell divides These terms will come up in RESEARCH PAPERS #1-5. Mitosis Mitosis is traditionally divided into five stages—prophase, prometaphase, metaphase, anaphase, and telophase —defined primarily on the basis of chromosome behavior as seen in a microscope. As mitosis is completed, the second major event of M phase—cytokinesis—divides the cell into two halves, each with an identical nucleus. Figure 17.3 of B Alberts, A Johnson, J Lewis, M Raff, K Roberts and P Walter “Molecular Biology of the Cell” 6th edition 2014, Garland Science Mitosis Interphase: Consists of G1, S, and G2 Prophase: Chromosomes condense, spindles are on opposite ends of the poles M Phase Mitosis Prometaphase: Nucleus is broken down, spindles start to attach Metaphase: Sister chromatids are at the midline of the cell (aligned on a plane known as the metaphase plate or equatorial plate), spindles have attached Note: There is a discrete stopping point between metaphase and anaphase. Division won’t progress until the spindles have attached to the sister chromatids and these chromatids are aligned at the equatorial plate. Progression is termed the ‘metaphase-to-anaphase transition.’ Anaphase: Sister chromatids separate to opposite poles Telophase: Sets of daughter chromatids arrive at the poles, nuclei begin to reform Cytokinesis: Division of the cytoplasm (begins in anaphase, is in process during telophase, and completes after telophase ends) Result: Two daughter cells, each with one nucleus. Read the details here Panel 17-1: The Principle Stages of M Phase (Mitosis and Cytokinesis) in an Animal Cell of B Alberts, A Johnson, J Lewis, M Raff, K Roberts and P Walter “Molecular Biology of the Cell” 6th edition 2014, Garland Science. Strategies to Exit The Cell Cycle Some cancer cells in tumor are in a reversible resting 1 stage known as G0 or the quiescent phase. In this 0 phase, cells remain metabolically active but do not proliferate. Cells in G0 are neither dividing nor preparing for division. These can be the majority! Most cells in adult animals enter the G0 stage of the cell cycle but resume proliferation as needed to replace cells that have been injured or have died (e.g., skin fibroblasts, smooth muscle cells, the endothelial cells that line blood vessels, and the epithelial cells of most internal organs). G Figure 17.4 of B Alberts, A Johnson, J Lewis, M Raff, K Roberts and P Walter “Molecular Biology of the Cell” 6th edition 2014, Garland Science Strategies to Exit The Cell Cycle G0 2 Differentiation A few types of differentiated cells are no longer capable of cell division 3 Figure 17.4 of B Alberts, A Johnson, J Lewis, M Raff, K Roberts and P Walter “Molecular Biology of the Cell” 6th edition 2014, Garland Science (e.g., cardiac muscle cells, red blood Apoptosis cells, neurons) Programmed cell death, this pathway is inactivated in many cancers What are the implications for cancer treatment that not all tumor cells are actively in the cell cycle at the same time? You may have to give a chemotherapy repeatedly, over an extended time to catch cells in a dividing, drug-sensitive stage It has been proposed that entering G0 enables cells to become resistant to anti-cancer compounds that target actively dividing cells For example, colorectal cancer cells can enter a G0-like state to survive chemotherapy. These cells are called drug-tolerant persisters (DTP) and are important drivers of therapy failure and tumor relapse Cell Cycle Control Figure 17.4 of B Alberts, A Johnson, J Lewis, M Raff, K Roberts and P Walter “Molecular Biology of the Cell” 6th edition 2002, Garland Science Progression through the cell cycle is tightly regulated There are 3 checkpoints/boundaries: 1. Between G1 and S phases The major checkpoint in mammalian cells Cells integrate external and internal signals (100s of them!) to answer a binary question: Is it appropriate to divide? 2. Between G2 and M phases Uses same cellular machinery as G1-S checkpoint Figure 17.4 of B Alberts, A Johnson, J Lewis, M Raff, K Roberts and P Walter “Molecular Biology of the Cell” 6th edition 2002, Garland Science 3. At the Metaphase-to-Anaphase transition Designed to ensure that all chromatids are attached to spindles Prevents daughter cells from having too many or too few Progression through the G1-S and G2-M checkpoints is regulated by cyclin-dependent kinases (Cdks) Kinase: An enzyme that phosphorylates another protein or DNA (the phosphate group comes from ATP) 2 major classes (based on identity of amino acid phosphorylated) Serine/threonine (Cdks are these) Tyrosine kinases (Also important in cancer, responsive to growth factors that trigger cell division. More on these later in the class.) Figure 17.4 of B Alberts, A Johnson, J Lewis, M Raff, K Roberts and P Walter “Molecular Biology of the Cell” 6th edition 2002, Garland Science Cyclin: A family of proteins that controls the progression of a cell through the cell cycle by activating Cdks First studied in yeast, where it was observed that their level changes (or cycles) during different stages of the cell cycle. The Cyclical Nature of Cyclins Figure 17.16 of B Alberts, A Johnson, J Lewis, M Raff, K Roberts and P Walter “Molecular Biology of the Cell” 4th edition 2002, Garland Science The Cyclical Nature of Cyclins Figure 17.11 of B Alberts, A Johnson, J Lewis, M Raff, K Roberts and P Walter “Molecular Biology of the Cell” 6th edition 2014, Garland Science There are multiple Cdk isoforms and multiple cyclins 3 different cyclinCdk complexes act in sequence to control the G1-S checkpoint A different cyclinCdk complex controls the G2-M checkpoint In addition to cyclin binding, Cdks are also regulated by phosphorylation Why go through this extra step? Why the increased complexity? Cdks produce profound changes. Cells need to make sure their activation is appropriate. Two independent regulatory processes need to occur concurrently to fully activate Cdks, cyclin binding and phosphorylation by CAKs. It’s about control (and signal integration). Cells are control freaks! Figure 17.12 of B Alberts, A Johnson, J Lewis, M Raff, K Roberts and P Walter “Molecular Biology of the Cell” 6th edition 2014, Garland Science In addition to cyclin binding, Cdks are also regulated by phosphorylation AND IT DOESN’T STOP THERE. Cells have many layers of protection to ensure Cdks aren’t activated inappropriately Don’t worry about names of these kinases but know that there are kinases that activate Cdks and there are kinases that inactive Cdks. AND there are phosphatases that can remove inhibitory phosphates from Cdks. How can a phosphate group at two different sites on a protein cause different effects (activate vs. inhibit)? They cause different conformational changes, different allosteric changes. Figure 17.12 of B Alberts, A Johnson, J Lewis, M Raff, K Roberts and P Walter “Molecular Biology of the Cell” 6th edition 2014, Garland Science So, what causes cancer? All cancers are due to a mix of: 1) Inappropriate expression of proteins that drive cell cycle progression (called oncogenes) AND 2) A loss of function of proteins that inhibit cell cycle progression (called tumor suppressors) Oncogene (derived from a proto-oncogene): A gene that has been inappropriately overexpressed or mutated and promotes progression through the cell cycle. – Proto-oncogenes are genes that cause normal cells to become cancerous when they are mutated. Proto-oncogenes are normal cellular genes that regulate cell growth and differentiation. Tumor suppressor: A gene whose expression inhibits cell cycle progression Is Wee1 kinase a protooncogene or a tumor suppressor? Is Cdc25 a proto-oncogene or a tumor suppressor? ~50% of all cancers have Cdc25 acting as an oncogene In addition to cyclins and phosphorylation, Cdks are also regulated by Cdk inhibitor proteins (CKIs) Are CKIs protooncogenes or tumor suppressors? DNA damage? Yikes! That can lead to mutations that give rise to oncogenes or inactivate tumor suppressors. This cell should not divide and potentially pass these mutations on to its progeny. p53 to the rescue! p53 is an Important Tumor Suppressor p53 is at very low levels in normal cells p53 is activated (phosphorylated) by DNA damage p53 is a transcription factor that will lead to the expression of CKI proteins and inhibit Cdks p53 will also start transcribing the genes that start apoptosis, giving the cell a finite amount of time to either fix the damage or undergo programed cell death Figure 17.12 of B Alberts, A Johnson, J Lewis, M Raff, K Roberts and P Walter “Molecular Biology of the Cell” 6th edition 2014, Garland Science p53 is inactivated in ~50% of cancers! Humans with one defective TP53 allele (the gene that encodes the p53 protein) have Li Fraumeni syndrome and a ~90% lifetime risk of many cancers, because they cannot properly shut down cells with DNA damage Peto’s Paradox Why doesn’t cancer risk scale with body size? Tollis, M., Boddy, A.M. & Maley, C.C. Peto’s Paradox: how has evolution solved the problem of cancer prevention?. BMC Biol 15, 60 (2017) Last regulatory mechanism (I promise): Proteolysis/degradation SCF is a ubiquitin ligase. It adds ubiquitin to CKIs to target them for degradation. Figure 17.15 of B Alberts, A Johnson, J Lewis, M Raff, K Roberts and P Walter “Molecular Biology of the Cell” 6th edition 2014, Garland Science So, what exactly do Cdk’s do? Turn on transcription! The key function of G1-Cdk complexes is to activate a group of transcription factors called the E2F proteins, which bind to specific DNA sequences in the promoters of a wide variety of genes that encode proteins required for S-phase entry, including G1/S-cyclins, Scyclins, and proteins involved in DNA synthesis and chromosome duplication. In the absence of mitogenic stimulation, E2F-dependent gene expression is inhibited by an interaction between E2F and members of the retinoblastoma protein (Rb) family. When cells are stimulated to divide by mitogens, active G1-Cdk accumulates and phosphorylates Rb family members, reducing their binding to E2F. The liberated E2F proteins then activate expression of their target genes Cdks phosphorylate Rb to allow E2F to transcribe genes required for S-phase transition Is Rb a protooncogene or a tumor suppressor? Figure 23.32 of B Alberts, A Johnson, J Lewis, M Raff, K Roberts and P Walter “Molecular Biology of the Cell” 4th edition 2002, Garland Science powerful proto-oncogene What kicks this whole process off? Signaling molecules! Mitogens are signals that act from outside the cell that induce a cell to begin cell division or enhances the rate of division. Learning Objectives Revisited 1. Describe how the cell cycle is regulated. There are 3 cell cycle check points. The major checkpoint in mammalian cells is at the G1-S boundary. The machinery that controls this transition are cyclin-dependent kinases (Cdks). Cdks are regulated by 1) cyclins, 2) activating phosphates, 3) inhibitory phosphates, 4) phosphatases that remove inhibitory phosphates, and 5) CKIs. 2. List what needs to ‘go wrong’ for a cancer to develop. 1) Inappropriate expression of proteins that drive cell cycle progression (oncogenes) 2) A loss of function of proteins that inhibit cell cycle progression (tumor suppressors) 3. Identify types of proteins could potentially be targeted in new cancer therapies. Lots! Use your imagination. p53 (gene therapy to deliver functional copies?), Cdks (hard to get a specificity but there are isoforms), Cdk regulatory proteins Homework Assignments for Jan 16 – Feb. 13 Complete Quiz #2 by Tuesday at 9:05 am. On Canvas. Two attempts. Download REVIEW ARTICLE #1: Hanahan, D., and R. A. Weinberg (2000). The hallmarks of cancer. Cell 100, 57-70. – Start reading and prepare to discuss during lecture on Feb. 1 Download RESEARCH PAPER #1: Druker at al. (1996). Effects of a specific inhibitor of the Abl tyrosine kinase on growth of Bcr-Abl positive cells. Nature Medicine 2, 561-564. – Start reading and prepare to discuss during lecture on Feb. 13