PHCL 4001 Lecture 2 Notes Spring 2024 PDF

PHCL 4001: MECHANISMS OF DRUG ACTION SPRING 2024 Lecture 2: The Cell Cycle LECTURE 1 (Thursday Jan. 18th) - Toward a greater target specificity, Part 1: The Cell Cycle Optional reading: Chapter 17 of Molecular Biology of the Cell, 5th edition, Bruce Alberts et al. (or the chapter on cancer biology w...

PHCL 4001: MECHANISMS OF DRUG ACTION SPRING 2024 Lecture 2: The Cell Cycle LECTURE 1 (Thursday Jan. 18th) - Toward a greater target specificity, Part 1: The Cell Cycle Optional reading: Chapter 17 of Molecular Biology of the Cell, 5th edition, Bruce Alberts et al. (or the chapter on cancer biology within different editions) The treatment of a cancer ultimately either directly or indirectly results in slowing or preventing unregulated progression through the cell cycle, and as a result understanding the cell cycle is essential to understanding the pharmacology underlying cancer treatments. Key Take-Away Questions: How is the cell cycle regulated? What needs to go wrong for a cancer to develop? What type(s) of protein could potentially be targeted in new cancer therapies? 2.1: Overview of the cell cycle The cell cycle involves the control of the accurate replication of the DNA and segregation of the chromosomes into two identical daughter cells. Most cells also double their mass and duplicate their cytoplasmic organelles in each cycle. Stages of the cell cycle: G1 phase - The interval between the completion of mitosis and the beginning of DNA synthesis. During this phase the cell monitors both its internal and external environments and commits to DNA replication and completion of the cell cycle only when conditions are appropriate. S phase - The phase during which the nuclear DNA is replicated. It takes-up about half the time of a typical mammalian cell cycle. G2 phase - The interval between the end of DNA synthesis and the beginning of mitosis. Allows the cell time to ensure that DNA replication is complete before progressing into mitosis. M - The process of 1) nuclear division (mitosis) and 2) cytoplastic division (cytokinesis). Note mitosis occurs relatively quickly. In a typical 24-hour mammalian cell cycle, only about one hour would normally be spent in M phase. 1) Highlights of each mitotic phase: Prophase - Chromosome condensation, spindles are on opposite ends of the poles Prometaphase - Nuclear membrane breakdown, spindle starts to attach Metaphase - Attachment of microtubules of mitotic spindle, alignment of chromosomes at equatorial plate Anaphase - Separation of sister chromatids to opposite poles Telophase – Daughter chromatids arrive at poles, chromosome de-condensation, reformation of intact nuclei. 2) Cytokinesis: Division of the cytoplasm (begins in anaphase, is in process during telophase, and completes after telophase ends) 1|Page PHCL 4001: MECHANISMS OF DRUG ACTION SPRING 2024 Lecture 2: The Cell Cycle 2.2: Exiting the cell cycle The standard cell cycle for fast growing mammalian cells is 24 hours but can be significantly longer. If extracellular conditions are unfavorable, cells can pause in their progress around the cycle and enter a specialized resting state called G0 (quiescent phase) that can last for years; most cells of the body are in this state and many will remain permanently in G0 until they or the organism dies. This state is reversible, as cells can enter the cell cycle again through G1. Most tumors have cells both actively moving through the cell cycle and in G 0. What is the significance of G0 to effectiveness of cancer therapy? Cells usually irreversibly exit the cycle when they differentiate or when they undergo apoptosis. What is the significance of irreversible differentiation to the treatment of some cancers? Significance of apoptosis to cancer and drug therapy? 2.3: Control of the cell cycle The cell cycle is normally highly regulated for the benefit of the organism as a whole: cells divide, differentiate, die, or enter a quiescent phase (G0) under the influence of extracellular signals. In most cells, there are checkpoints in the cell cycle at which the cycle can be arrested if previous events have not been completed or if the extracellular environment is not favorable. In higher eukaryotes, extracellular signals that arrest the cycle usually act at the G1 control point. If extracellular conditions are favorable and the appropriate signals are present, cells in G1 or G0 progress through this checkpoint and are committed to DNA replication. If DNA is not replicated properly or is damaged, the cycle pauses at the G2 checkpoint, preventing mitosis from occurring until the problem is fixed. The third major check point occurs at the metaphase-to-anaphase transition. Mitosis is halted at this checkpoint until all the chromosomes are attached properly to the spindle. The proteins that control the cell cycle first appeared over a billion years ago, and they are highly conserved among eukaryotes. As a result, model organisms like yeast and xenopus have been highly insightful in studying mammalian cell cycle control. 2.4: Nature of the checkpoints Kinases. A kinase is an enzyme that phosphorylates a protein or a nucleic acid. The source of the phosphate is usually from the -phosphate of ATP. It has been estimated that approximately 1/3 of the eukaryotic proteins are phosphorylated at any given time and that 2% of the human genome encodes kinases. Phosphorylation is one of the major mechanisms through which signal transduction pathways are turned on/off. Cyclins activate cyclin-dependent kinases (Cdks). The concentration of cyclins change during the cell cycle. Activation of the Cdks can result in the activation/deactivation of other kinases, the initiation of DNA replication, the activation/deactivation of transcription factors, depolymerization of the nuclear lamina, and the targeting of numerous other proteins for degradation. Each of the different cyclin-Cdk complexes serves as a molecular switch that triggers a specific cell-cycle event. The cyclins not only activate their Cdk partners but also direct them to specific target proteins. Cdks can be further activated 2|Page PHCL 4001: MECHANISMS OF DRUG ACTION SPRING 2024 Lecture 2: The Cell Cycle by Cdk-activating kinases (CAKs) that phosphorylate near the entrance to the substrate binding site. Alternatively, phosphorylation at other sites can inhibit Cdk activity. Some negative regulator proteins function as tumor suppressors in that they inhibit uncontrolled passage through the cell cycle. Tumor suppressor genes are mutated or deleted in most cancers. Significance of tumor suppressors to cancer therapy? p53 prevents cells with damaged DNA from being replicated and, if the damaged DNA cannot be repaired, the protein will trigger apoptosis (a programmed cell death). This protects the organism from the propagation of cells with mutations that could eventually lead to cancer. p53 is inactivated in over half of all human cancers. An oncogene is a mutated gene that has the potential to cause cancer. Before an oncogene becomes mutated, it is called a proto-oncogene, and it plays a role in regulating normal cell cycle progression. Ubiquitinases. Ubiquitin-targeted degradation is a major mechanism through which the levels of cyclins and other key cell cycle regulatory proteins are regulated during the cell cycle. A ubiquitin-ligase enzyme complex attaches multiple copies of ubiquitin to specific sequences, and this targets the protein for complete destruction in proteasomes. There are two major classes of ubiquitin-ligase complexes involved with cell cycle regulation: The SCF complex. The SCF complex is required for ubiquitylation and destruction of CKIs and other proteins prior to passage through the G1-S checkpoint. Phosphorylation of specific amino acids of specific proteins targets them for the SCF-catalyzed reaction. 2.6: Passing through a checkpoint Changes in transcription can dramatically alter the level of some cyclins. In animal cells, the transcription of G1cyclins is stimulated by extracellular signals. The retinoblastoma (Rb) protein is a tumor suppressor that acts by sequestering E2F, which is a powerful transcription factor that promotes the transcription of several cyclins as well as other proteins required for progression through the G1 restriction site. Upon stimulation by extracellular signals, active G1-Cdk accumulates and phosphorylates Rb. The phosphorylation reduces the affinity for E2F and allows E2F to act as a transcription factor. c-myc is a powerful proto-oncogene. Over expression or inappropriate expression of c-myc can result in a gain of function and is a major driving force of many cancers. The mutant overactive form or inappropriately expressed form of these genes are called oncogenes; contrast these with tumor suppressors, which are a loss of function that contributes to most cancers. Positive feedback loops amplify the activation of the CdK-cyclins and sharpen the transition through the different cell-cycle restriction points. 3|Page