OS 201 Human Cell Biology Cell Cycle and Cell Death PDF

OS 201: HUMAN CELL BIOLOGY CELL CYCLE AND CELL DEATH UPCM 2029 | Dr. Paul Medina | LU3 A.Y. 2024-2025 organelles and raw ingredients....

OS 201: HUMAN CELL BIOLOGY CELL CYCLE AND CELL DEATH UPCM 2029 | Dr. Paul Medina | LU3 A.Y. 2024-2025 organelles and raw ingredients. OUTLINE It must grow in size and double the cellular components. I. Cell Cycle E. Proto-oncogenes and Typically when cell leaves the cell cycle, it exits while still in G1 A. Types of Cells Tumor Suppressor ○ Mammalian cells often spend 8-10 hours in G1 B. Cell Cycle Phases Genes A cellular decision is made that causes the cell to be arrested in C. Main Purpose of Cell III. Cell Death Mechanisms G thus it will enter G0 state Cycle A. Cell Death Restriction point D. Somatic Cell Division B. Apoptosis ○ Phase within G1 where the cell decides if it will continue for E. Mitosis C. Apoptotic Pathway another round of cell cycle. II. Cell Cycle Regulation D. Other Cell Death ○ If the cell passes through this point, the cell is now committed to A. Cell Cycle Length Mechanisms the cell cycle B. Cell Cycle IV. References ○ Otherwise, it will go to G0 Checkpoints S-PHASE C. Signal Molecules D. CDK Regulation S phase is usually 6-10 hours DNA is replicated (S is Synthesis) ○ Each of the 46 chromosomes is duplicated by the cell I. CELL CYCLE ○ Not necessarily the number of chromosomes double, but the A. Types of Cells genetic material doubles because of DNA replication Labile Cells G2-PHASE ○ Cells that constantly undergo division and have a high turnover G2 is usually 4-6 hours ○ Found in tissues that need high rate of cell renewal or tissue that Cell growth continues in preparation for mitosis quickly regenerates (i.e., epithelial cells, hematopoietic cells, Main concern is to check if S Phase proceeded without mistake (i.e., germ cells) if everything went right during the S phase) Stable Cells ○ The cell “double checks” the duplicated chromosomes for error, ○ Cells that do not divide under normal circumstances making any needed repairs ○ Often quiescent (arrested in the G0 phase), but can be stimulated ○ If everything is in order, the cell will ready itself for another round to divide of mitosis is nuclear division, while cytokinesis is cellular division ○ Able to re-enter the cell cycle and proliferate in response to injury When mitosis and cytokinesis are combined, we refer to is as or certain signals (i.e., hepatocytes, renal tubular cells, fibroblast, cell division endothelial cells) ○ Duration of cell division varies based on cell type Permanent Cells In humans, usually takes 24 hours ○ Cells that have permanently exited the cell cycle Some cell types take less than 8 hours to divide, others over a ○ Does not divide under normal physiologic conditions year ○ Cannot regenerate or proliferate post-injury (with some ○ Variability in duration is usually due to the G1 phase. exceptions such as skeletal muscle fiber satellite cells), and Other phases are pretty much stable in length of duration typically results in scarring ○ Damage is often irreparable (i.e., neurons, cardiomyocytes, G0-PHASE skeletal muscle fibers) Cell cycle arrest Some cardiomyocytes have very limited regenerative capacity, ○ The cell decides to leave the cell cycle but cannot proliferate ○ Technically, when the cell leaves the cell cycle, it exits while in G1 B. Cell Cycle Phases C. Main Purpose of Cell Cycle It is a series of events that takes place in a cell as it grows and To accurately transmit genetic information divides ○ From the mother cell to the two resulting daughter cell It has 2 phases: To maintain normal ploidy (i.e., diploidy) ○ Interphase (non-dividing state) ○ Humans have diploid cells It is the longer phase ○ Ploidy: the number of set chromosomes in a cell or in the cell of Can be divided into G1, S, and G2 an organism ○ M-phase D. Somatic Cell Division Comprises both mitosis and cytokinesis Either mitosis (somatic cells) or meiosis (zygomatic cells), but Interphase - 95% of cell cycle (longest phase) mainly mitosis ○ Organelle duplication, DNA replication, growth occurs here Mitosis is mainly involved in cytokinesis (cellular division ○ 3 phases: proper) G1 - Gap 1 Cytokinesis is between the M-phase and G1 phase. S - Synthesis It is in the border of M-phase and interphase G2 - Gap 2 M-phase - Mitotic phase + Cytokinesis ○ Also refers to meiosis G1-PHASE Metabolically active Organelle duplication, but no DNA replication Duration is variable - shorter in embryonic and cancer cells; ○ Embryonic cells divide fast similar to cancer cells with the latter also being uncontrollable Prepares for S phase or DNA replication ○ Cell that remain in G1 for a long time = G0 (permanent tissues, such as neural cells or bone cells) For highly differentiated cells Figure 1. Overview of Cell Cycle G-PHASE Cell grows in size ○ Cellular contents, excluding the chromosomes, are duplicated ○ During this phase, the cell just finished cell division thus, it is relatively small. If it decides to undergo another cycle of cell division, it needs to grow big because right now (at this stage), it only has a few Figure 2. Restriction Point Trans #1 TG#29: Tiam-Lee, Timbulan, Tolentino, P., Tolentino, V., Uy, F., Uy, M., Valimento TH: Ilagan 1 of 9  R-POINT Chromosomes and 4 Sister Chromatids After cytokinesis, each daughter cell contains 2 chromosomes Restriction point Between the M-phase up to the R-point, the cell is very sensitive to M PHASE MUTATIONS mitogenic growth factors, and TGF-β Euploidy ○ TGF-β suppresses cellular division ○ Additions of whole chromosome sets (n, 2n, 3n, 4n) Once the cell is through the R-point, even with the presence of ○ Can occur in tissues that have differentiated, such as TGF-β, the cell is committed to go through the cell cycle. hepatocytes, cardiomyocytes, and bone marrow It is also in this phase where the cell leaves the cell cycle and Aneuploidy go to G0 ○ Additions or subtractions of single sets of chromosomes (2n+1, Pathway is dictated by phosphorylation of Retinoblastoma Protein, 2n-1, 2n+2…) which is phosphorylated by activated Cyclin-dependent Kinase 4 ○ Seen in disease such as Down Syndrome (Trisomy 21), and 6 Klinefelter’s Syndrome (XXY), Turner’s Syndrome (only 1 X Phosphorylated Retinoblastoma Protein unbinds from E2F chromosome) transcription factors, and free E2F activates transcription of genes After cytokinesis, each daughter cell contains 2 chromosomes for S-phase progression. Cyclin D+Cdk4/6 + Pi/PO4 -> pRB-PO4 -> E2F Release -> CYTOKINESIS AND CLEAVAGE FURROW Transcription of crucial S-phase genes S-PHASE Synthesis phase Cell is committed to cell division once it enters S Phase DNA and centrosome replicate ○ DNA replication is semi-conservative The DNA double helix separates, with each strand being paired up with a newly synthesized complementary strand to produce two new DNA molecules; two identical daughter genomes Two daughter DNA = one old, one new daughter DNA During DNA replication, one side of replication is continuous, the other side is discontinuous After the S phase, 46 chromosomes are still present (not 92) ○ DNA content (genetic material) doubles, not the number of Figure 5. Cytokinesis in Plants and Animals chromosomes ○ Two identical daughter genomes are produced Not a phase of mitosis Occurs when a cleavage furrow forms and splits the two daughter cells apart Slightly overlaps the M Phase, and occurs at early anaphase Cleavage furrow is formed by a contractile ring that separates cytoplasm between two cells, pinching off and splitting cytoplasm into two Contractile ring is comprised of various cytoskeletal proteins ○ Anillin works as a scaffold for myosin, actin, and septin filaments It functions to stabilize the cleavage furrow in cell division ○ Actin is the main cytoskeletal protein involved in pinching the cell and binding of microtubules ○ Septin acts as a cellular scaffold and a barrier to prevent diffusion of other molecules between the two forming daughter cells At the furrow, PIP2 is accumulated in the membrane, as well as anillin (binding actin, myosin, and septins) Pleckstrin Homology domain of anillin (PH) binds with septin and reacts with PIP2 Figure 3. Phases of a cell cycle G2-PHASE Main goal: check whether the S phase has proceeded correctly ○ Checks for and repairs errors in chromosome duplication ○ If no error is found, cell goes through another round of mitosis Cell continues to grow Phase where the stage at which the cell is in can be determined ○ Cells at different stages of the cell cycle can also be distinguished by their DNA content DNA replication has already occurred in G2 → thus, it has twice the amount of DNA content compared to G1 92 chromatids are present, with 46 chromosomes Figure 6. Cleavage furrow formation with actin and septins E. MITOSIS Takes up 5% of the entire cell cycle Composes both Mitosis and Cytokinesis ○ Mitosis without cytokinesis results in a multinucleated cell ○ Mitosis with cytokinesis results in two similar separate daughter cells Consists of 5 Stages: ○ Prophase ○ Prometaphase ○ Metaphase ○ Anaphase ○ Telophase Sister chromatids are considered individual chromosomes after separation Figure 7. Cleavage furrow formation including all specific cytoskeletal proteins involved MULTINUCLEATED HUMAN CELLS Figure 4. Chromosomes throughout the Cell Cycle Some cells in the human body are exceptions in that a single cells At the G1 phase, there are 2 Chromosomes and 0 Sister Chromatids can contain multiple nuclei In the G2 phase, genetic material is doubled, resulting in 2 ○ Osteoclast OS 201 Cell Cycle and Cell Death 2 of 9  ○ Hepatocytes Cell remains in G2 phase while DNA repair is done ○ Straited Muscles ○ Multinucleated giant cells (MGCs) in urinary bladder M Checkpoint II. CELL CYCLE REGULATION Between metaphase and anaphase Anaphase is blocked if chromatids are not properly assembled in A. CELL CYCLE LENGTH the mitotic spindle during metaphase Varies by cell type All chromosomes must be properly attached to mitotic spindle via ○ Can last by 8-10 hours to more than a year TF4/kinetochore[2026 Trans] ○ Shorter: embryonic cells, stem cells, sperm cells ○ Or else it could lead to the unequal distribution of G1 Phase chromosomes between two daughter cells or missegregation ○ Most varied in length (vs. other phases of cell cycle) ○ If they are not properly attached, the cell cycle is arrested ○ Growing mammalian phase often spend 8-10 hours in G1 phase Error results in anaphase block; mitosis stops ○ Determines cell cycle length and cellular decision is made here whether the cell will go to G0 or proceed to the cell cycle C. SIGNAL MOLECULES ○ Prolonged in stable or permanent cells (called G0) KINASE Cells in prolonged G1 may be mistaken as being in the G0 phase ○ Rapid or non-existent in rapidly dividing cells An enzyme that phosphorylates proteins by transferring a G2 phase phosphate group from a high energy molecule (e.g. ATP) to an ○ Usually shorter than G1; usually 4-6 hours amino acid residue of the protein S phase Named after their substrates ○ Usually 6-10 hours 2 major types ○ Ser/Thr Kinase B. CELL CYCLE CHECKPOINT ○ Tyr Kinase Control points where stop and go signals regulate the process These amino acids have hydroxyl groups that serve as substrate Checkpoints check for DNA or genome integrity for phosphorylation ○ To serve one of its main purposes of transmitting genetic CYCLIN-DEPENDENT KINASE (CDK) information accurately Retinoblastoma protein (Rb) Inactive unless bound to a cyclin molecule ○ Regulates cell cycle, particularly at G1/S checkpoint CDK + cyclin = active ○ Releases E2F transcription factors when phosphorylated (active ○ E.g. Maturation Promoting Factor (MPF) = CDK-Cyclin complex form) and binds to them when unphosphorylated (inactive) CDKs exert their effects by phosphorylating specific serine and/or When Rb releases E2F, genes necessary for DNA synthesis and threonine residues on their target proteins responsible for various S phase entry are activated cell cycle events [2025 Trans] When Rb binds to E2F, it prevents the expression of genes Phosphorylation induces conformational changes that alter their required for DNA replication catalytic activity and interaction with other proteins [2025 Trans] ○ Phosphorylated by the binding of cyclin and CDK CDK will have active kinase activity once it binds with a cyclin, and phosphorylates the target protein (Figure 7.) ○ The target protein will perform a new function due to being phosphorylated ○ Note that phosphorylation can activate or inactivate the function of proteins Figure 8. Checkpoints for the different phases of the cell cycle Additional Information [Trans 2027] Restriction (R) Point Point in G₁ phase where the cell commits to the cell cycle to continue into the S phase or exit and go to G0 Gives an alternative route → G0 Figure 9. Phosphorylation of CDK to change the activity of target protein ○ Depends on the status of the DNA material CYCLIN Growth factor dependent ○ Mitogenic GFs allow progress through cell cycle Was first identified in sea urchins ○ TGF-β prevents cell from getting past the R point CDK is non-functional without cyclin Mnemonic: M = maaari (Mitotic GFs), T = tigil (TGF-β) Was first called Maturation Promoting Factor (MPF) but now defined Cell goes to S or G₀ phase depending on which growth factor it is as the CDK-cyclin complex [2026 Trans] sensitive to Cyclin concentrations rise in G2 and fall during mitosis Regulated by cyclin dependent kinases ○ Named cyclin because of fluctuations in concentrations throughout the cell cycle G1/S Checkpoint ○ Begin accumulating in S phase DNA damage checkpoint ○ Degraded during mitosis Entrance to S phase is blocked if genome is damaged ○ MPF activity peaks before each cell division [2026 Trans] Decides if cell will be quiescent or proceed to cell cycle CDKs are not broken down so MPF concentration reflects the ○ Whether the cell should divide, delay division, or enter resting amount of cyclin stage Nutrition dependent[2025 Trans] ○ Nutrients and enzymes required for DNA synthesis must be present S Checkpoint DNA damage checkpoint DNA replication is halted if genome is damaged There can be errors during replication which causes the cell to halt in S phase Figure 10. Cyclin Fluctuation Concentration. Cyclin begins accumulating late in the S G2/M Checkpoint phase and falls during M phase. Located at the boundary between G2 and M phase Table 1. Common Cyclins [Based on 2028 Trans] Entrance into M phase is blocked if DNA replication is incomplete Cyclin Description ○ Cell cannot initiate mitosis Cyclin D Comprises 3 related proteins (D1, D2, D3) ○ Genome must also be accurate before cell can divide OS 201 Cell Cycle and Cell Death 3 of 9  Binds CDK4 and CDK6 Cyclin D1 binds CDKs early to mid G1 and together with Cyclin E/CDK2 inactivate pRB (cell cycle inhibitor Functions in the restriction point Associated with Rb Binds to CDK2 Cyclin E/CDK2 phosphorylates p27 (Cyclin D Cyclin E inhibitor) which tags it for degradation Functions in G1 to S phase transition Promotes expression of Cyclin A S-phase cyclin Binds CDK2 and CDK1 Functions in G1/S and G2/M transition Cyclin A Key regulator of cdc25 and CDK1 Involved in activating Cyclin B/CDK1 complex Associated also with entry to the M phase just like Cyclin B Figure 12. CDK Regulation by Polyubiquitinationi First to be discovered (where the MPF term came from) PHOSPHORYLATION Mitotic cyclin Mainly by allowing the substrate-binding site of the kinase to be Cyclin B Binds CDK1 to make MPF available to the substrate Functions in entry to M phase Phosphorylation status of cyclin and cyclin-dependent kinase Associated with the transition from G2 to M complex dictates its activity ○ Phosphorylated: inactive; Dephosphorylated: active However, depending on the location of the phosphorylation, it can sometimes cause the complex to be active ○ Phosphorylation creates a conformational change on the kinase, allowing the substrate-binding site to open up and be made available to the substrate Figure 11. Cyclin Expression Cycle. Different cyclins rise in concentration per cell cycle phase. SENESCENCE VS. REPLICATIVE SENESCENCE Senescence ○ A general term where the cells that typically have the capacity to divide loses that ability Replicative senescence ○ Same as senescence but mostly because of shortened Figure 13. CDK Regulation by Phosphorylation telomeres BINDING TO CDK INHIBITORY PROTEINS (CKIs) D. CYCLIN-DEPENDENT KINASE REGULATION CKIs binding to CDK complex will inhibit the kinase activity Two general classes CYCLIN SYNTHESIS AND DESTRUCTION ○ Inhibitors that immediately inactivate the complex upon binding Since CDKs are cyclin-dependent, controlling the amount of cyclin to it, even if the complex is still intact present can also control whether they become active or inactive Example: p21 family of CDK inhibitors Cyclin synthesis mainly depends on transcription and translation ○ Inhibitors that disassemble the cyclin and cyclin-dependent Cyclin destruction occurs through polyubiquitination kinase complex, rendering it inactive Ubiquitination Example: INK4 family of CDK inhibitors Polyubiquitination of a protein means targeting it for destruction Applicable not only for cyclins, but for many different proteins Additional Information [Trans 2028] throughout the cell p21 family Mechanism: ○ p21, p27, p57 ○ Target protein gets polyubiquitinated and then sent to INK4 family proteasome ○ p15, p16, p18, p19 ○ Proteasome breaks down the protein into short peptides ○ Inhibits by replacing cyclin ○ Mutation of inhibitor CDK-Cyclin complex allows cells to Additional Information [Trans 2028] continually divide and disassemble complex ○ p16 iis the most frequently mutated in human tumors CDK Regulation by Polyubiquitination CKIs regulate the G1-S transition Mitotic cyclin destruction box ○ Section in cyclin protein sequence involved in degradation ○ Sites for polyubiquitination that allows cyclins to be escorted to proteasomes Figure 14. CDK Inhibitors E. PROTO-ONCOGENES AND TUMOR-SUPPRESSOR GENES Proto-oncogenes Genes that have a normal function and are involved in the progression of the cell cycle ○ These allow the cell cycle to continue OS 201 Cell Cycle and Cell Death 4 of 9  Mutations in proto-oncogenes, usually a gain of function Table 2. Cell Death Mechanisms in Humans mutation, cause it to dysregulate the progression of the cell cycle Become oncogenes when mutated Programmed Unprogrammed Other Forms of Cell Death Cell Death Cell Death Oncogenes Mutant forms of proto-oncogenes Apoptosis Necrosis Ferroptosis Unlike proto-oncogenes that can be switched on and off, Autophagy (ACD) Parthanatos oncogenes remain continuously active Pyroptosis Entosis Driver for unregulated cell division Necroptosis ○ Accelerates cell growth and division; can lead to cancer There are two copies (from fraternal and maternal sources); * ACD - Autophagic Cell Death mutation in one copy is enough for the cell to divide uncontrollably B. APOPTOSIS The nature of the mutation is dominant ○ Normal proto-oncogene may also exist but is overridden by the Type of programmed cell death mutant proto-oncogene (oncogene) Essential for the following processes: ○ Normal development Tumor Suppressor Genes ○ Tissue homeostasis Genes that stop the cell cycle when expressed ○ Elimination of damaged or unwanted cells ○ Normally happens when there are damages detected in the DNA; Highly regulated process characterized by specific morphological tumor suppressor genes act and stop the cell cycle and biochemical changes There are two copies (from fraternal and maternal sources); despite mutation in one copy, the other copy can still function normally FEATURES OF APOPTOSIS ○ Two-hit hypothesis Energy-dependent process ○ During the first hit, when one allele gets mutated, there’s still one ○ Active; requires a lot of ATP functioning allele Regulated and controlled ○ However, if both alleles get mutated, tumor suppressor genes can ○ Intrinsic pathway - initiated by the mitochondria no longer stop the cell cycle even if there are damages in the DNA ○ Extrinsic pathway - initiated in death receptors in cell membrane material Morphologic changes in cell There is loss of heterozygosity ○ Cell shrinkage The nature of the mutation is recessive ○ Membrane blebbing ○ Chromatin condensation F. CLINICAL CORRELATES OF THE CELL. CYCLE ○ Nuclear fragmentation Cancer Phagocytosis ○ Uncontrolled cell proliferation ○ There is formation of apoptotic bodies From bypassing cell cycle checkpoints ○ NO creation of damage-associated molecular patterns (DAMPs); ○ Genetic instability as such, it is non-inflammatory Mutations in genes that regulate cell cycle Tumor suppressor genes (e.g. p53) MORPHOLOGICAL CHANGES IN THE CELL Oncogenes (e.g. Ras) 1. Nucleus will condense (pyknosis) and blebbing of the plasma ○ Therapeutic targets membrane. Overall, the cell shrinks in size. CDK and PARP Inhibitors that interfere with cell cycle 2. The nucleus will also become fragmented (karyorrhexis), releasing checkpoints or DNA repair pathways chromatin. Eventually the blebs will pinch off, forming apoptotic Neurodegenerative Diseases bodies. ○ Neurodegeneration 3. Apoptotic bodies will be engulfed by phagocytic cells. Disruptions in cell cycle contribute to neurodegenerative diseases such as Alzheimer’s disease and Parkinon’s disease ○ Cell death Dysregulation of the cell cycle can lead to increased cell death particularly in neurons Aging ○ Cellular senescence Cells age, enter senescence, and stop dividing but remain metabolically active Lead to aging-related diseases due to problems in tissue repair and regeneration Tissue repair and regeneration ○ Wound Healing Cells proliferate to repair damaged tissues ○ Stem Cell Therapy Cell cycle regulation is vital in controlling the differentiation and proliferation of stem cells Potential applications in regenerative medicine Additional Information [Trans 2028] Alvocidib ○ Also known as Flavopiridol Figure 15. Morphological changes in the cell during apoptosis. ○ Flavonoid alkaloid CDK9 kinase inhibitor under clinical development for treatment of acute myeloid leukemia Two forms of nuclear dissolution is observed in apoptosis Hallmarks of Cancer ○ Pyknosis - nuclear shrinkage ○ Incidences where cancer can be promoted ○ Karyorrhexis - nuclear fragmentation Self-sufficiency in growth signals Additional Information (2028 Trans) Evading apoptosis and immune surveillance ○ Karyolysis - nuclear fading Insensitivity to anti-growth signals Another form of nuclear dissolution Sustained angiogenesis Chromatin dissolution due to action of DNAses and RNAses Tissue invasion and metastasis DNA damage stress limitless replicative potential Oxidative stress Proteotoxic stress Metabolic stress III. CELL DEATH MECHANISMS A. CELL DEATH Human body maintains a constant number of cells ○ Mechanisms exist for ensuring other cells in the body are removed, when appropriate (e.g., injured/damaged cells) Average cell death numbers: ○ Adult: 50-70 billion cells die daily ○ Child (8-14 y/o): 20-30 billion cells die daily There are many different ways that the cells could die Figure 16. Pyknosis versus karyorrhexis. ○ The table shows only the major cell death mechanisms ○ There are still other minor mechanisms as cellular death is Specific Occurrences cell-specific and context dependent A number of activities take place Cell death is a very complex phenomenon ○ Occupation of death receptors → dimerization of Bcl-2 family members → release of cytochrome C → activation of Caspases → OS 201 Cell Cycle and Cell Death 5 of 9  activation of DNases was supposed to be played during the lecture but was not Translocation of phosphatidylserine discussed. ○ Normally, this phospholipid is located at the inner membrane ○ During apoptosis, it is trafficked to the extracellular side ATP-dependency Internucleosomal DNA fragmentation ○ Nuclease destroys DNA between nucleosomes ○ Shows ladder pattern when run on a gel NO apoptosis at +4°C NO inflammation MECHANISM OF APOPTOSIS Extrinsic Pathway Activated by death ligands, TNF, FasL, and other molecules Can recruit the intrinsic pathway through Bid molecules Intrinsic Pathway Activated by bacterial toxins, O2 radicals, and other factors Eventually, it finds its way into the mitochondrion Will NOT necessarily recruit the extrinsic pathway Figure 18. Role of trophic factor in apoptosis Caspases Proteins employed by apoptosis which degrade other proteins Made as inactive precursors - procaspases Activated by other proteins when the right signal is received One caspase is involved in nuclear dissolution ○ Cleaves the lamin proteins (which stabilize the nuclear structure) resulting in irreversible breakdown of nuclear membrane Table 5. Caspases in Apoptosis Type of Caspase Enzyme Caspase 2 human & mouse Caspase 8 human & mouse Initiator Caspase 9 human & mouse Caspase 10 human only Caspase 3 human & mouse Executioner Caspase 6 human & mouse Caspase 7 human & mouse Figure 17. Extrinsic versus intrinsic apoptosis pathway Additional Information (from Lecture Slides) Table 3. Steps in the Extrinsic and Intrinsic Pathway Note: The following table shows the caspases in Pyroptosis, another form of programmed cell death. This is NOT the focus of Extrinsic Pathway Intrinsic Pathway the lecture. 1. External ligands (e.g., death 1. Bacterial toxins, O2 radicals, Table 6. Caspases in Pyroptosis ligands, TNF, FasL, etc.) binds and other molecules activate to a death receptor (e.g. Bim/Puma proteins Type of Caspase Enzyme Fas-associated protein with 2. These activates Bcl-2/Bcl-xL Caspase 1 human & mouse death domain [FADD]) proteins which interact with 2. FADD (an adaptor protein) Bax/Bak proteins. Caspase 4 human activates Caspase 8 3. Bax/Bak proteins permeabilize Inflammatory Caspase 5 human 3. Caspase 8 activate Caspase 3* the mitochondrial outer 4. Caspase 3 activates other membrane by forming a pore Caspase 11 mouse Caspases downstream (e.g., 4. Cytochrome C exits the Caspase 12 mouse & some humans Caspase 6 and 7). mitochondria; then interacts 5. Eventually, there is proteolysis with Apaf1+ and activates of proteins involved. Caspase 9 FACTORS THAT INFLUENCE APOPTOSIS 5. Caspase 9 activates Caspase 3 Withdrawal of positive signals ○ Growth factors for neurons * Caspase 8 from extrinsic pathway can recruit the intrinsic ○ Interleukin-2 (IL-2) - pro-inflammatory cytokine pathway by cleaving Bid to form truncated Bid (tBid). This will Receipt of negative signals then interact with Bax/Bak proteins in the mitochondrial ○ Increased levels of oxidants within the cell - oxidative stress membrane. ○ Hypoxia Table 4. Summary of Extrinsic versus Extrinsic Pathway ○ Radiation ○ Chemotherapy Feature Extrinsic Pathway Intrinsic Pathway ○ Damage to DNA by oxidants Initiating External death signals Internal stress (DNA ○ Death activators: tumor necrosis factor-alpha (TNF-⍺), Signal (FasL, TNF, TRAIL, etc.) damage, oxidative stress, lymphotoxin (TNF-𝛽), Fas ligand (FasL) etc.) ROLE OF APOPTOSIS IN THE BODY Key Players Death receptors (Fas, Bcl-2 family proteins TNFR, adapter proteins (Bax, Bak, Bcl-2) In Development [FADD]) Apoptosis has a role in development ○ Formation of fingers and toes of fetus Mitochondrial Optional (thru Bid Essential (MOMP, Fetus starts with webbed hands but these interdigit- areas die Involvement cleavage and MOMP) cytochrome C release) ○ Sloughing off of the inner lining of the uterus (menstruation) Caspases Caspase-8 (initiator), Caspase-9 (initiator), ○ Formation of proper connections between neurons in brain Involved Caspase-3 (effector) Caspase-3 (effector) Regulation Regulated by external Regulated by cellular signals and immune stress and survival signals system Inflammation Non-inflammatory unless Non-inflammatory, no cell excessive (triggered by lysis necroptosis) Additional Information (from Lecture Slides) Note: The following figure is a representation of the animation that OS 201 Cell Cycle and Cell Death 6 of 9  ○ Ischemic injury can trigger apoptosis in affected tissues leading to tissue damage and organ dysfunction C. AUTOPHAGY A cellular process involving the degradation and recycling of cellular components Often described as the cell’s “self-eating” mechanism STAGES OF AUTOPHAGY Initiation ○ Activated by cell stress ○ Formation of the phagophore around the cellular components destined for degradation Elongation Figure 19. Foot of mouse embryo. Acridine orange (AO) staining of mouse embryo ○ Expansion of the phagophore, engulfing the target material footplates between 12.5 and 14.5 days of development reveals cell death (bright green) in the interdigital tissue of the developing limb (A–A’’). Corresponding stage Maturation limbs stained with F4/80 reveal macrophages (brown) in the same location as they ○ Phagophore matures into an autophagosome, a engulf the resulting apoptotic corpses (B–B’’). Adapted from Wood & Martin, 2017. double-membrane vesicle containing the cellular debris Fusion Control of Cell Numbers and Cell Size ○ Lysosome and autophagosome fuses, forming the autolysosome Three processes operate to control the eventual form a body part where degradation occurs takes: cell growth, cell division, and cell death Degradation Single-celled organisms grow as fast as they are able to, limited by ○ Lysosomal enzymes break down the cellular components within factors such as food availability and waste the autophagosome into their building blocks On the other hand, multicellular organisms receive from other cells Recycling in the body (not just from nutrition) ○ Recycled materials can be used to generate energy or to ○ e.g., when growing cells in a culture, these would divide until they synthesize new cellular components start touching each other; cell communication for contact inhibition to prevent overcrowding ○ Cancer cells don’t have contact inhibition, thus forming crowded cell clusters Classification of typical signals from neighboring cells: ○ Mitogens - allow cell to enter the cell cycle ○ Growth factors - increase cell mass ○ Survival factors - suppress apoptosis Uses of Apoptosis in the Body Many processes in the body make use of apoptosis ○ Growth of embryo ○ Insulin-dependent diabetes ○ Tissue homeostasis ○ Atherosclerosis ○ Immunology ○ Myocardial infarction Figure 20. Stages of Autophagy ○ Chronic viral diseases ○ AIDS ○ Neurodegenerative diseases ○ Development and treatment ○ Reperfusion injury of malignancies Apoptosis is used to destroy cells ○ Cells infected with viruses ○ Cells of the immune system after an immune response ○ Cells with DNA damage - potentially cancerous if not killed ○ Cancer cells CLINICAL CORRELATES Cancer Tumorigenesis ○ Defects in apoptotic pathways can contribute to the development of cancer ○ Cancer cells often evade apoptosis, allowing them to proliferate uncontrollably Cancer Therapy ○ Inducing apoptosis in cancer cells is a major goal of many cancer treatments like in chemotherapy and radiation therapy Figure 21. Stages of Autophagy under the electron microscope Neurodegenerative Diseases IMPORTANCE OF AUTOPHAGY Alzheimer’s Disease Cellular Quality Control ○ Dysregulated apoptosis of neurons contributes to ○ Removes damaged organelles and proteins, preventing their neurodegeneration and cognitive decline accumulation and potential harm to the cell ○ It was found that brains of deceased individuals affected by the Adaptation to Stress disease had shrunk due to apoptosis of brain cells ○ Helps cells survive by providing nutrients (e.g., hypoxia or Parkinsons’ Disease starvation) ○ Increased neuronal apoptosis in the substantia nigra leads to the ○ Recycled materials become source of nutrition loss of dopamine-producing cells Protection Against Disease Huntington’s Disease ○ Protect against various diseases, including cancer, ○ Mutant huntingtin protein triggers apoptosis in neurons, leading neurodegenerative disease, and infectious diseases to progressive neurodegeneration CLINICAL CORRELATES Autoimmune Disease Systemic Lupus Erythematosus (SLE) Neurodegenerative Diseases ○ Inappropriate apoptosis of lymphocytes can lead to the release of Alzheimer’s Disease autoantigens and the development of autoimmune responses ○ Autophagy dysfunction contributes to the accumulation of Rheumatoid arthritis amyloid-beta plaques and tau protein tangles, hallmarks of ○ Dysregulated apoptosis of immune cells can contribute to Alzheimer’s disease chronic inflammation and joint damage Parkinson’s Disease ○ Autophagy impairment leads to the accumulation of misfolded Viral infections alpha-synuclein protein, a key player in Parkinson’s disease Viral Replication Huntington’s Disease ○ Some viruses, such as HIV, can evade the immune system by ○ Defects in autophagy can exacerbate the accumulation of inhibiting apoptosis of infected cells mutant huntingtin protein, contributing to neurodegeneration HIV becomes “residents” and latent inside the cells. Cells become a reservoir of the virus because they prevent the cell Cancer from undergoing apoptosis. Tumorigenesis Viral-Induced Cell Death ○ Autophagy can acts as both a tumor suppressor and a tumor ○ Certain viruses can induce apoptosis in infected cells as a promoter defense mechanism Inhibit tumorigenesis by removing damaged organelles and proteins Ischemic Injury Promote tumor cell survival and metastasis under certain Heart Attack and Stroke conditions OS 201 Cell Cycle and Cell Death 7 of 9  Cancer Therapy CAUSES OF NECROSIS ○ Inhibiting autophagy can enhance the efficacy of chemotherapy and radiation therapy Physical injury ○ Trauma, burns, or extreme temperatures Metabolic Diseases Chemical injury Type 2 Diabetes ○ Exposure to toxins or harmful chemicals ○ Autophagy plays a role in insulin sensitivity and glucose Infections metabolism ○ Bacterial or viral infections ○ Dysfunctional autophagy can contribute to insulin resistance and Ischemia the development of type 2 diabetes ○ Lack of blood supply deprive cells of oxygen and nutrients Obesity Immune-mediated injury ○ Autophagy is involved in regulating lipid metabolism NECROSIS VS APOPTOSIS ○ Impaired autophagy can lead to lipid accumulation and obesity Aging Cellular Senescence ○ Autophagy declines with age, contributing to cellular senescence and aging-related diseases Lifespan ○ Enhancing autophagy has been shown to extend lifespan in model organisms AUTOPHAGIC CELL DEATH (ACD) Autophagy is primarily a cell survival mechanism. In certain situations however, it can lead to cell death. This is called autophagic cell death. Switch from Survival to Death Intensity and Duration of Stress ○ Prolonged or severe stress can overwhelm the cell’s capacity for autophagy-mediated survival, triggering cell death Impaired Autophagic Flux ○ Defects in the autophagic machinery, such as mutations in autophagy-related genes, can lead to accumulation of autophagic vesicles and cell death Cross-talk with Apoptosis ○ Autophagy can interact with apoptotic pathways ○ Autophagy may suppress apoptosis, while in others, it may promote cell death by activating apoptotic signaling pathways Mechanisms of Autophagic Cell Death Excessive Autophagy ○ Overactive autophagy can deplete essential cellular components, Figure 23. Comparison between Necrosis and Apoptosis leading to cell death Autophagic Flux Blockage ONCOSIS ○ Impaired autophagic flux can result in the accumulation of Pre-lethal stage that often precedes necrosis autophagosomes and toxic cellular debris, ultimately leading to “Pre-necrotic” stage cell death Apoptosis Induction ○ Autophagy can trigger apoptosis by activating apoptotic signaling pathways or by releasing pro-apoptotic factors from autophagosomes Figure 24. Oncosis vs Apoptosis Figure 22. Representative cases of autophagic cell death (ACD) NECROSIS VS ONCOSIS Autophagic cell death can occur in midgut of drosophila, during insulin withdrawal, tumor crisis, cerebral ischemia reperfusion Table 7. Necrosis vs Oncosis (mitophagy), and Chronic Restraint Stress (CRS) Feature Necrosis Oncosis D. NECROSIS Regulated Unregulated Can be regulated by Form of cell death that occurs when cells are subjected to severe certain conditions stress or injury Unlike apoptosis (programmed cell death), necrosis is an Cell Swelling Significant Significant uncontrolled process (unprogrammed cell death) that can damage Membrane Loss of membrane Membrane integrity may surrounding tissues integrity integrity be maintained initially FEATURES OF NECROSIS Inflammatory Significant Less pronounced Cell swelling response ○ Cell inside the cell swells due to the influx of water Cellular fate Cell lysis and release of Can lead to necrosis or In apoptosis, there is shrinkage cellular contents apoptosis Organelle swelling ○ Organelles within the cell also swell (mitochondria, ER) Loss of membrane integrity ○ Cell membrane loses its integrity eventually causing cellular lysis, leading to the leakage of cellular contents into the extracellular space Inflammation ○ Release of cellular contents triggers an inflammatory response Apoptosis and autophagy do not induce inflammation OS 201 Cell Cycle and Cell Death 8 of 9  Figure 25. Necrosis vs Oncosis CLINICAL CORRELATES OF NECROSIS Ischemic Injury Myocardial infarction ○ Heart attack, where heart muscles die due to lack of oxygen Stroke ○ Brain cell death due to lack of blood flow Peripheral arterial disease ○ Tissue death in the extremities due to poor blood circulation Infections Bacterial infections ○ Bacterial infections can lead to tissue necrosis, particularly in cases of necrotizing fasciitis (gangrenous) Viral infections ○ Some viruses can induce cell death (e.g., hepatitis or viral encephalitis) Fungal infections ○ Fungal infections, especially those caused by opportunistic fungi, can lead to tissue necrosis Autoimmune Diseases Systemic lupus erythematosus (SLE) ○ Autoimmune attacks can lead to tissue damage and necrosis Rheumatoid arthritis ○ Inflammation and immune cell infiltration can cause joint damage and necrosis Neurodegenerative Diseases Neurodegenerative disorders ○ Diseases like Alzheimer’s and Parkinson’s can involve neuronal necrosis Trauma Physical trauma ○ Injuries can cause tissue damage and cell death Cancer Tumor necrosis ○ Chemotherapy and radiation therapy, can induce necrosis in tumor cells E. ADDITIONAL DETAILS In any pathological state, cells can die in multiple ways (not exclusively apoptosis, ACD, or necrosis) ○ Different ways of cell death can occur simultaneously even in the same type of cell and environment The cause of this phenomenon is still unknown In translational application, apoptotic cell death is preferred since it is non-inflammatory ○ Necrosis is inflammatory and can be problematic as it may induce senescence and further cellular damage You want your medications to be non-inflammatory as much as possible A lot of diseases are inflammation-based, most likely chronic F. ZOMBIE GENES Cell death does not necessarily follow the organismal death in a multicellular organism Myocardium, pericardial fluid and blood from thirty cadavers in relation to post-mortem interval showed a few genes active and still being expressed after 12 hours (Villanueva, 2013) In mice, 515 genes were seen kicking into gear, and were functioning at full capacity up to 24 hours after death In zebrafish, 548 genes retained their function for 4 whole days after the animals had died before showing any signs of winding down A. CITATION Wood, W., & Martin, P. (2017). Macrophage Functions in Tissue Patterning and Disease: New Insights from the Fly. Developmental Cell, 40(3), 221–233. doi:10.1016/j.devcel.2017.01.001 OS 201 Cell Cycle and Cell Death 9 of 9