BSR 2.04 - Cell Death and Degeneration - v1_08.28.23.pdf

BASIC SCIENCE RESEARCH Cell Death and Degeneration Block 2 Mark Pierre S. Dimamay, MD | September 13, 2024...

BASIC SCIENCE RESEARCH Cell Death and Degeneration Block 2 Mark Pierre S. Dimamay, MD | September 13, 2024 Trans 2.04 B. Apoptosis v OVERVIEW I. Cell Death IV. Condition Associated with Programmed cell death A. Necrosis Defective Apoptosis ”Cells are altruistic in the sense that they can B. Apoptosis Regulation sacrifice themselves for the sake of other cells” C. Comparison: A. Two General Avenues Cells shrink and develop bubble-like blebs → form Apoptosis vs Necrosis for Disease vesicles that are easy to clean up II. Techniques in Studying B. Inhibitors of Apoptosis Required for normal development and protection Apoptosis Proteins (IAPs) ○ Ex. development of digits, the formation of the A. Apoptotic Parameters C. Apoptosis-based B. Morphological Therapeutics in lens of the eyes, cells of the immune system features (Microscopy) Clinical Trials undergo cell death if impaired C. DNA Fragmentation “Clean” cell death (Figure 1L) D. Alterations of the Cell ○ Condensation, Fragmentation (including nucleus Membrane and nuclear DNA), & the formation of smaller III. Mechanisms of Apoptosis apoptotic bodies → packed into vacuoles → A. Intrinsic Pathway engulfed by macrophages B. Extrinsic Pathway Involves the breakdown of mitochondria with the C. Caspase Cascade LEGEND release of Cytochrome C : Important information Phosphatidylserine is found only in the inner : Good-to-know info from lecturer membrane leaflet of the cytoplasm (Figure 2) : Supplementary/Background Info ○ When this part undergoes apoptosis, it flips to the : Exception outer leaflet (using the flippase enzyme) ○ This can be a marker of apoptosis (to identify ABBREVIATIONS which cells are undergoing apoptosis) PS Phosphatidylserine AIF Apoptosis Inducing Factor HMW High Molecular Weight LMW Low Molecular Weight TUNEL TdT-mediated dUTP nick end labeling TdT Terminal Deoxynucleotidyl Transferase dNTP Deoxynucleotide Triphosphate LEARNING OBJECTIVES Characterize types of cell death Learn the techniques in studying cell death Figure 2. PS translocation from the inner membrane of the lipid bilayer to the Describe the mechanism of apoptosis outer membrane side Correlate cell death with diseases Apoptosis as a Normal Physiological Process I. CELL DEATH Apoptosis is a part of normal/proper development ○ Resorption of tadpole tails A. Necrosis ○ Formation of human fingers and toes Cell death due to mechanical damage (ex. Exposure Membranes in between are eliminated via to toxic chemicals) or other environmental factors apoptosis Characteristic features: ○ Proper networking of neurons ○ Cell & organelle swelling Apoptosis eliminates potentially damaging cells ○ Leakage of cell contents ○ Cells infected with viruses are programmed to die ○ Inflammation of surrounding tissues ○ Cells of the immune system “Messy” type of cell death (Figure 1R) ○ Cells with irreparable DNA damage ○ Cells rupture → cytoplasmic contents leak and ○ Cancer cells scatter to neighboring tissues → immune response triggered (ex. inflammation) Table 1. Examples of apoptosis in development ” In surgery, when you cut a person open, you Fate of Cell SIGNAL RECEIVED actually cause the death of a lot of cells” Lens cells ○ Forms during embryonic development Eye ○ Consists of apoptotic cells that have replaced the innards with the clear protein crystallin Figure 1. Cell death photomicrograph. (Left) Apoptosis (Right) Necrosis Page 1 of 10 | TH: BAUTISTA, M | TF TG 5 | CASTILLO, FLORENTINO, GRINO Checked and verified: TP LIM, A. GAMAD, MJ  BSR 2.04 Cell Death and Degeneration Table 2. Summary of differences between apoptosis and necrosis APOPTOSIS NECROSIS Normal physiologic process Anoxia Lack of growth Physical Causes factors damage Hormonal Chemical influences damage Mild toxic influences First apparent swelling no Shrinking or Swelling Figure 3. Close-up View of Lens Cell. Apoptosis forming cell changes Convolution the lens of the eye Condensation Cells of the villi pynknotic nucleus Segmentation kardowhexis ○ Villi are small finger-like projections Nuclear changes DNA Mangolipis - of the intestinal wall fragmentation Intestines ○ Cells arise at the base of villi → (ladder-like in gel travel to the tip over a few days → electrophoresis) eventually die → sloughed off to be Surface replaced by new cells protrusions Cell membrane Smoothing Skin cells Budding (off) changes Lysis ○ Begin life in its deepest layers → changes in PS migrate to the surface in layers → distribution Skin undergo apoptosis along the way → Mitochondrial Swelling - resulting dead layer is what will changes Rupture form the protective outer layer Active changes (epidermis) in gene T-lymphocytes (T-cells) expression Metabolic or ○ T-cells are white blood cells that (e.g. Bcl, Bax) Synthetic - are critical components of the Active protein changes immune system synthesis ○ These mature in the thymus gland Protease ○ Ineffective T-cells or those that are activation self-reacting are programmed to die Table 3. Additional differences on Apoptosis vs. Necrosis Thymus before they have the chance to [Robbins] enter the bloodstream Clinical significance: Apoptosis by self-reactive T-cells is a way to prevent autoimmune diseases Cells of the uterine wall Uterus ○ Die and slough off during menstruation Cells infected by viruses or those that sustain irreparable genetic mutations undergo apoptosis Others Clinical significance: Failure of these (Protective) mutated cells to undergo apoptosis may lead to cancer Apoptosis vs Necrosis Apoptosis vs. Necrosis can be discriminated by looking at: ○ Cell morphology ○ Cell DNA ○ Expression at certain genes (present at apoptosis, absent in necrosis) Page 2 of 10 | TH: BAUTISTA, M | TF TG 5 | CASTILLO, FLORENTINO, GRINO  BSR 2.04 Cell Death and Degeneration II. TECHNIQUES IN STUDYING APOPTOSIS Advances in apoptosis research include discovery of new apoptosis detection methods. Figure 6. DAPI stain of the nuclear components of cells. Apoptotic cells are indicated by red arrows which have condensed nuclei. B. DNA Fragmentation Figure 4. Apoptotic parameter timeline A. Morphological Features of Apoptotic Cells using Microscopy Figure 7. Nucleosomal banding pattern DNA is broken into small pieces, through physiological (apoptosis) or external processes ○ Another approach to detect apoptosis by analyzing genomic DNA content of cells DNA fragmentation is one of the hallmarks of apoptosis ○ Properly extracting (not physically shearing) DNA from the apoptotic cell reveals that the degradation is not random ○ DNA is cut at regular intervals between nucleosomes → cut at “linker regions” Results in fragments that are multiples of Figure 5. Microscopic images of apoptotic cells depicting different stages of around 180-200 base pairs in length apoptosis: (A) shrinkage, (B) nuclear fragmentation (C) apoptotic bodies (D) membrane blebbing. Microscopy is the most basic tool of apoptotic cell research The morphology of apoptotic cells are easily seen via microscope ○ Able to identify the different stages of apoptosis (e.g. nuclei condensing, fragmentation, apoptotic body formation, etc.) ○ Cells with membrane blebbing signify they are undergoing apoptosis DAPI Staining in Fluorescence Microscopy Using a dye/stain can help further visualize the stained nuclei of cells and their structures via fluorescence microscopy. ○ Can differentiate normal cells from those Figure 8. Example of a DNA ladder of an apoptotic cell (lane 3) and a normal apoptosis cell (lane 2). Note the predominant HMW shown in lane 2. Apoptotic processes target specific regions of DNA cleavage via endonuclease activation. Page 3 of 10 | TH: BAUTISTA, M | TF TG 5 | CASTILLO, FLORENTINO, GRINO  BSR 2.04 Cell Death and Degeneration ○ Resulting in DNA fragments with varying C. In Situ Apoptosis Detection by TUNEL Method molecular weights TdT-mediated dNTP nick end labeling (TUNEL) Appears as → Distinct DNA ladder after gel ○ TdT: terminal deoxynucleotidyl transferase electrophoresis ○ dNTP: deoxynucleotide triphosphate DNA extraction is important because if the cell was Newer method of identifying apoptotic cells that is physically ruptured (e.g. shearing), then the points of done while in culture cleavage would be random. ○ ↑ DNA fragmentation in apoptosis leads to: ○ The DNA ladder would look like a white ↑ DNA strand breaks smudge/smear. ↑ Labeling and staining in TUNEL method DNA ladders can be useful in differentiating TUNEL Method detects DNA fragmentation by apoptotic cells from normal and necrotic cells. labeling 3’ ends of a DNA strand, which is extended by an X-dNTP that is tagged by a fluorescent Table 4. DNA Fragmentation Facilitated by Physical Injury vs. Enzymes molecule By Enzymes By Physical ○ “Nick end labeling” - tagging ends of DNA that (Apoptosis) Injury (Necrosis) have been cut DNA Not Random Random Fragmentation Specific, clear sizes where Size of DNA nucleases cut No specific sizes DNA and fragment them DNA Ladder Present Absent Distribution of DNA in Normal and Apoptotic Cells There are distinct differences between the high molecular weight (HMW) and low molecular weight (LMW) DNA between normal and apoptotic cells ○ HMW DNA → Long strands of DNA found in Figure 9. Labeling Principle with TUNEL mechanism on the right healthy, non-apoptotic cells ○ LMW DNA → Shorter, fragmented pieces of DNA TUNEL Mechanism produced when HMW DNA is cleaved at apoptosis Table 5. DNA Fragmentation Facilitated by Physical Injury vs. Enzymes Compart Normal Cell Apoptotic Cell ment HMW LMW HMW LMW Nucleus + (-) ↓ ↑ Cytoplasm (-) (-) (-) ↑ Figure 10. Non-apoptotic cell (left) and apoptotic cell (right) using TUNEL method showing fluorescent signal (in green). In an apoptotic cell, there is ↑ DNA fragmentation in In normal cells: apoptosis due to endonuclease activity ○ ↑ Increased HMW and (-) Absent LMW in the ○ Means more DNA strands are available for nucleus X-dNTP tagging Both DNA and nuclear membrane are fully ○ The more DNA fragments, the more free 3’ ends intact that are labeled ○ (-) Absent HMW and LMW in the cytoplasm Visible under the microscope No DNA fragmentation occurs in healthy cells Density can measured In apoptotic cells: Hence, TUNEL assay of apoptotic cells: ○ ↓ HMW and ↑ LMW in the nucleus ○ Shows a stronger fluorescent signal Fragmentation caused by activation of ○ Can be visualized under a microscope endonucleases Can differentiate normal and apoptotic cells in ○ (-) Absent HMW and ↑ LMW in the cytoplasm culture without the need to harvest As apoptosis progresses and the nuclear membrane breaks down, the fragmented LMW DNA is released into the cytoplasm Page 4 of 10 | TH: BAUTISTA, M | TF TG 5 | CASTILLO, FLORENTINO, GRINO  BSR 2.04 Cell Death and Degeneration Figure 11. The same monolayer of cells with non-specific stain (left) and specific stain with TUNEL assay (right). Non-specific Staining Figure 12. Cells receive many types of signals that may trigger different types ○ Cannot differentiate between apoptotic and of responses. If there is no signal, cells may die. non-apoptotic cells. Specific Staining with TUNEL Assay Table 6. Fate of cells depending on signal/s received ○ Apoptotic cells in the same monolayer are easily Fate of Cell SIGNAL RECEIVED identifiable and can be differentiated from SURVIVE If given the correct signals (blue) normal cells If addition of additional signals will Apoptotic index is determined by: DIVIDE trigger the cells to proliferate (red) ○ Manual counting, or through use of AI software If addition of signals may also cause them to differentiate from a DIFFERENTIATE 𝐴𝑝𝑜𝑝𝑡𝑜𝑡𝑖𝑐 𝑖𝑛𝑑𝑒𝑥 = # 𝑜𝑓 𝑓𝑙𝑢𝑜𝑟𝑒𝑠𝑐𝑒𝑛𝑡 𝑐𝑒𝑙𝑙𝑠 & 𝑏𝑜𝑑𝑖𝑒𝑠 progenitor cell to a differentiated cell 𝑇𝑜𝑡𝑎𝑙 𝐶𝑒𝑙𝑙𝑠 (green) If all signals coming from other cells D. Significance of Apoptotic Parameters DIE are withdrawn leading to the A good anti-cancer drug candidate is able to apoptotic pathway discriminately trigger apoptosis in cancer cells while not harming healthy cells E.g. Challenge: Grow cells first from a biopsy. ○ Apoptotic parameters can be used to test and ○ This applies in offering a service to cancer patients show this where we can get the tumor & isolate cells from it, and subsequently do in vitro cytotoxicity testing to III. MECHANISMS OF APOPTOSIS identify which chemo drug will work on the cells What makes a cell decide to end its life? and use it to treat the patient ○ The withdrawal of positive signals for continued Prevents trial and error in the patient survival There are 2 general mechanisms by which cells “No cell is an island, they need positive signals undergo apoptosis: from neighboring cells” ○ Intrinsic Pathway Positive signals are needed for continued ○ Extrinsic Pathway survival, important to keep cells viable E.g. Intrinsic factors, cell adhesion A. Intrinsic Pathway ○ Receipt of negative signals Generated by signals arising within the cells, i.e. cell E.g. Death activators, radiation, viral infection, stress or DNA damage cytotoxic T-cells, DNA damage ○ Effective DNA repair mechanisms are present ○ Lack of any signal (cell isolation) ○ But as cells age, there are certain biological If cells are too far from adjacent cells, they pathways that slow down and become inefficient, would not receive signals which accumulate When trying to grow cells in a culture, they ○ E.g. oxidative stress, free radicals, affected must be seeded with a defined minimum mitochondrial levels (low ATP levels) seeding density (number of cells needed to be ○ Damage causes apoptosis placed on the dish for viable growth) ↪ Cells are usually kept at 10,000 cells per cm 2 Page 5 of 10 | TH: BAUTISTA, M | TF TG 5 | CASTILLO, FLORENTINO, GRINO  BSR 2.04 Cell Death and Degeneration Through DNA Replication In this manner, p53 is able to maintain cellular and genetic stability. Clinical significance: In cancer, usual genes that mutate are usually p53 genes. If p53 is active, it would cause the death of cancer cells due to their mutations. However, if p53 is mutated, cancer cells do not die. This knowledge can provide information on cancer treatments. The p53 mutation is repaired. If not able, a vector can also be used. Such as what is found on vaccines, that carries p53 and transfect the tumor with an active p53 and cause cell death in cancer cells. (ex. Drugs such as Gendicine) Figure 13. Intrinsic pathway of apoptosis from DNA replication Through the Release of Cytochrome C The intrinsic pathway can be triggered in DNA replication ○ This is by the shortening of telomeres (ends of chromosomes) at the lagging strand after every cell division Telomeres protect the actual coding regions When a nuclear DNA undergoes replication, it does not matter if the telomeres shorten as long as the actual genes are protected[2027 Trans] ○ The shortening of telomeres will eventually Figure 15. Intrinsic Pathway of Apoptosis from the Release of Cytochrome C. affect coding sequences which could cause DNA damage and subsequently mutations Another intrinsic process is the release of cytochrome One way of measuring cell senescence & aging C from the mitochondria which can result in the ○ To prevent this from happening, a trigger is activation of Caspase-9, an initiator of the caspase activated, also known as the Hayflick limit, once cascade that facilitates apoptosis. the telomeres get too short, to activate apoptosis. Cell activates the tumor suppressor gene, p53, B. Extrinsic Pathway for the cell to go through apoptosis if it incurs irreparable damage. TP/TH/TG Note: This section was not discussed by Dr. Dimamay Tumor Suppressor Protein p53 but was included in the PPT. Other factors that cause DNA damage can also trigger apoptosis. This pathway is regulated by a Triggered by cell death activators binding to tumor suppressor protein known as p53. receptors at the cell surface. ○ Help the immune system identify cells that are affected or damaged, which may affect the entire system so they are eliminated Triggered by dangerous reactive oxygen species ○ Fas ligand (FasL) ○ Turns Necrosis Factor-alpha (TNF-α) Figure 14. Pathways for DNA Damage. p53 is activated by DNA damage (Figure 14). ○ If the damage can be repaired, p53 arrests the cell = cycle to allow DNA repair and restarts the cell Figure 16. Extrinsic pathway of apoptosis from the binding of Fas ligand to Fas protein cycle once repair is done. The extrinsic pathway is initiated by a signal ○ But if the damage is severe, p53 will trigger originating extracellularly. oto eliminate the damaged cell. apoptosis Binding of a Fas ligand (from another cell) to the Fas Page 6 of 10 | TH: BAUTISTA, M | TF TG 5 | CASTILLO, FLORENTINO, GRINO  BSR 2.04 Cell Death and Degeneration protein (a receptor) will trigger apoptosis by C. Caspase- Cysteine Proteases activating Caspase-8 O in the cell carrying the Fas protein (Figure 16). Because Fas is able to trigger apoptosis in the target cell, it is considered as a death activator, together with other molecules like TNF-α. TP/TH/TG Note: The following points were lifted from the 2027 Trans. Figure 18. Caspase Cascade Caspase Cascade Caspases: induces apoptosis in the cell and cleaves Asp residues of the AA chain Two main classes of caspases Figure 17. Extrinsic pathway of apoptosis from TNF-α. ⑤ Of ○ Initiator caspase: Caspase -8 and -9 Upstream activators of the effector caspases ○ Effector caspase: Caspase -3, -6, and -7 Another signal that activates the extrinsic pathway - - - Executioners of the cell comes from death activators like TNF-α. Cleaves proteins that actually induces These death activators fall and intersect with the apoptosis in the cell caspase activation. Role of Caspases in Apoptosis Figure 17. Extrinsic and Intrinsic pathway (combined) mechanisms for Figure 19. Role of Caspases in Apoptosis apoptosis. Extrinsic signals from the outside include survival Caspases link the intrinsic and extrinsic signals factors, growth factors, death activators, chemokines, Most signals converge in the effector caspases (-3, -6, etc. -7) Intrinsic signals which may come from the ○ Activation of these effector caspases lead to cell mitochondria include Caspase-9 and Cytochrome C. shrinkage, DNA fragmentation, and DNA repair Apoptosome: required to be formed in order to make caspases ○ APAF 1 → Apoptosome → Caspase-9 ○ Location of apoptosome: Caspase-9 Page 7 of 10 | TH: BAUTISTA, M | TF TG 5 | CASTILLO, FLORENTINO, GRINO  BSR 2.04 Cell Death and Degeneration IV. CONDITIONS ASSOCIATED WITH DEFECTIVE Overexpression of bcl-2 → increased APOPTOSIS REGULATION androgen → survival and proliferation of Apoptosis is required to maintain the equilibrium prostate cancer cells → death and integrity of the cell Therapeutic potential: downregulation of bcl-2 Defects on the regulation of apoptosis can be → androgen depletion → apoptosis of prostate caused by overregulation or underregulation cancer cells → cancer control → prolonged survival A. Two General Avenues for Disease Autoimmunity: failure to eliminate autoreactive Aberrant Apoptosis Activation lymphocytes Normal cells that should not die or undergo Persistent infections: failure to eradicate bacteria or apoptosis are abnormally activated → normal cells virus-infected cells die continuously Examples: B. IAPS: Inhibitors of Apoptosis Proteins ○ AIDS: trigger cell death of healthy lymphocytes Functions to keep caspases in check ○ Neurodegenerative diseases: neurons undergo Contains at least one (1) copy of a Baculovirus IAP apoptosis due to certain factors repeat domain Parkinson’s disease, Huntington’s disease, Suppresses apoptosis when overexpressed Alzheimer’s disease, stroke and epilepsy ○ Some IAPs directly bind and inhibit caspases ○ Ischemic injury Can be a potential drug target for cancer IAP genes can be used to treat neurological diseases Excessive Apoptosis Ischemia: Reperfusion injury (stroke, myocardial C. Apoptosis - based Therapeutics in Clinical Trials infarction) The market has a lot of clinical trials aimed at ○ In addition to apoptotic cell death, necrosis is also discovering both anti- and pro-apoptotic strategies, present. Heart failure: Loss of myocardiocytes depending on the specific disease and therapeutic Neurodegeneration: Loss of dopamine-secreting target cells and other neurons Table 7. Apoptosis-based Clinical Drug Trial ○ Parkinson’s disease, Huntington’s disease, Drug or Apoptosis Disease Alzheimer’s disease, Amyotrophic lateral sclerosis Therapeutic Target Effect Indicators Inflammation Target Osteoarthritis: Chondrocyte depletion Core Apoptosis Targets Human Immunodeficiency virus: Depletion of T lymphocytes IDN6556 Caspases Anti Acute Infection Bacterial Infections: Apoptosis-inducing virulence Arthritis, proteins secreted into the cytosol of host cells VX-740 Caspase-1 Anti Inflammation ○ Example: Binding to and activation of Caspase-1 Ipa proteins of Shigella Genasense BCL2 Pro Cancer Sip B proteins of Salmonella Inputs into Apoptosis Machinery YopJ protein (SUMOylase) of Yersinia ○ Binding to and activation of death receptors from TRAIL DR4, DR5 Pro Cancer the TNF family PPARγ,, Cadd protein of Chlamydia CDDO Pro Cancer IKK ○ Allograft rejection and graft versus host disease ○ Type 1 Diabetes α5β3- Vitaxin Pro Cancer Immune-mediated destruction of the islets of integrin Langerhans α5β3- EMD121974 Pro Angiogenesis Impaired Apoptosis integrin Cells that should undergo apoptosis continue to live E1A Gene E1A Pro Cancer Examples: Therapy ○ Cancer: Normal cells should undergo apoptosis ONYX-015 Unknown Pro Cancer when their DNAs are damaged INGN201 p53 Pro Cancer (NSCL) ○ Autoimmune disorders: lymphocytes that recognize self-antigens should be eliminated, If Neuroprotection these lymphocytes live, autoimmune disorders can CGP3466B GAPDH Anti (Parkinson’s occur. Disease) ○ Viral infections PDE5A Exisulind Pro Cancer (Colon) (PKG) Insufficient Apoptosis Neuroprotection Cancer: abnormal cell accumulation Memantine NMDAR Anti (Alzheimer’s, ○ Resistant to radiation, chemotherapy, and Dementia) immunotherapy SAHA HDAC Pro Cancer ○ Example: Prostate Cancer Page 8 of 10 | TH: BAUTISTA, M | TF TG 5 | CASTILLO, FLORENTINO, GRINO  BSR 2.04 Cell Death and Degeneration A. Initiator Caspase B. Caspase-9 REFERENCES C. Effector Caspase Mark Pierre S. Dimamay, MD. Notes from Cell Death D. - Death Receptors and Degeneration CONDITIONS ASSOCIATED WITH DEFECTIVE BOOKS APOPTOSIS REGULATION Moore, K.L., Dalley, K.F., Agur, A.M. (2017) 8. Which of the following is NOT TRUE about Aberrant Apoptosis Activation? REVIEW QUESTIONS A. Includes diseases like Cancer and Autoimmunity - B. Normal cells undergo abnormal apoptosis CELL DEATH C. Includes diseases like Heart failure and neurodegeneration 1. What is known as a messy cell death? D. Cells that should undergo apoptosis proceeds to A. Apoptosis cell death B. Necrosis - C. Caspases *** End of Review Questions *** D. Death Receptor Pathway TG Note: All questions with “*” are lifted from 2027 2. Which of the following is FALSE for necrotic Trans processes? A. Expression of genes - RATIONALE B. Can be caused by anoxia C. Mitochondria swells and ruptures D. Can be caused by physical or chemical damage 1. B. Apoptosis is “Clean cell death” TECHNIQUES IN STUDYING APOPTOSIS 2.A [Recall] 3. Under microscopy, what signifies that there is an 3. C [Recall] ongoing apoptosis? A. Inflammation 4. D, As mentioned, apoptosis undergo clean cell death which - B. Accumulation of neutrophils mean they are programmed to undergo non-random DNA C. Membrane blebbing fragmentation D. Membrane rupture 5. A [Recall] 4. What is TRUE in DNA fragmentation by enzymes in apoptosis? 6. C Extrinsic pathway is also known as death receptor pathway A. Random DNA Fragmentation B. Absent DNA Ladder 7. C [Recall] - C. No specific size of DNA D. Non-random DNA fragmentation 8. A [Recall] 5. Which of the following is TRUE for an apoptotic cell under TUNEL mechanism? SUMMARY What is the difference between apoptosis and necrosis? A. -Endonucleases are activated which cuts DNA Table 2. Summary of differences between apoptosis and necrosis into smaller fragments APOPTOSIS NECROSIS B. Apoptotic cells have a few 3’ ends for labeling Normal C. Apoptotic cells are not visible under a physiologic microscope process Anoxia D. Density can’t be measured Lack of growth Physical Causes factors damage MECHANISMS OF APOPTOSIS Hormonal Chemical influences damage 6. Which of the following is TRUE about the Extrinsic Mild toxic Pathway? influences A. Triggered by signals rising within the cell ~ First apparent Shrinking or B. Involves the release of Cytochrome C Swelling cell changes Convolution C. Triggered by death receptors Condensation D. Targets Asp residues Segmentation Nuclear changes DNA - 7. What are the executioners in the cells that cleave fragmentation protein and induce apoptosis? (ladder-like in gel electrophoresis) Page 9 of 10 | TH: BAUTISTA, M | TF TG 5 | CASTILLO, FLORENTINO, GRINO  BSR 2.04 Cell Death and Degeneration Surface ○ Apoptotic cells can’t be protrusions quantified Cell membrane Smoothing Budding (off) TUNEL method changes Lysis changes in PS ○ Labeling principle: 3’ ends of distribution DNA strand is extended by an Mitochondrial Swelling X-dNTP → tagged by - changes Rupture fluorescent molecule Active changes ○ Step 1: Cells on slide in gene ○ Step 2: Add TdT and expression FITC-dUTP Metabolic or ○ Step 3: Fluorescence or light (e.g. Bcl, Bax) Synthetic - microscopy Active protein changes ○ Apoptotic index: total number synthesis Protease of TUNEL stained fluorescent activation cells versus the total number of unstained cells Table 3. Additional differences on Apoptosis vs. Necrosis [Robbins] Flow cytometry: once PS goes to the outer membrane leaflet, an antibody binds to PS can be used and be tagged Cell Membrane by a fluorescent probe alterations Quantifies how many cells are going to apoptosis and how many are not What are the mechanisms of Apoptosis? Intrinsic pathway: from signal inside the cells which induces release of Cytochrome C and occurs during DNA replication Extrinsic pathway: from signals outside the cell which usually come from death activators like Fas ligand, TNF-a, and lymphotoxin Both the intrinsic and extrinsic pathway converge in the caspase cascade which would lead to cell death Correlate cell death with diseases Defective apoptosis regulations cause numerous What are the fates of cells depending on the signals received? conditions Table 4. Fate of cells depending on signal/s received With aberrant apoptosis activation, cell death is Fate of Cell SIGNAL RECEIVED excessively activated. This can be seen in SURVIVE If given the correct signals conditions like neurodegenerative disorders, HIV/AIDS, and ischemia If addition of additional signals will Impaired apoptosis causes insufficient cell death. DIVIDE trigger the cells to proliferate Cells that are supposed to die continue to live. This If addition of signals may also cause is usually seen in cases of cancer, autoimmune DIFFERENTIATE them to differentiate from a disorders, and persistent infection progenitor cell to a differentiated cell If all signals coming from other cells DIE are withdrawn leading to the apoptotic pathway What are the techniques in studying cell death? Apoptotic Techniques and signs of apoptosis Parameter Microscopy: Membrane blebbing Morphological DAPI staining in fluorescence features of microscopy apoptotic cells ○ INCREASE stain intensity ○ DECREASE nucleus size Agarose gel electrophoresis ○ DNA ladder DNA ○ Specific & clear sizes of DNA Fragmentation where nucleases have cut them Page 10 of 10 | TH: BAUTISTA, M | TF TG 5 | CASTILLO, FLORENTINO, GRINO  BASIC SCIENCE RESEARCH Cell Death and Degeneration Block 1 Mark Pierre S. Dimamay, PhD | August 18, 2023 BSR 2.04 OVERVIEW “Clean” cell death (Fig. 1L) I. Cell Death IV. Conditions Associated with ○ Condensation, fragmentation (including nucleus and A. Necrosis Defective Apoptosis nuclear DNA), and the formation of smaller apoptotic B. Apoptosis Regulation bodies → packed into vacuoles → engulfed by C. Apoptosis vs Necrosis A. Two General Avenues for macrophages II. Techniques in Studying Disease Apoptotic bodies have intact membranes that aid in Apoptosis B. IAPs: Inhibitors of A. Apoptotic Parameters Apoptosis Proteins their elimination via phagocytosis B. Morphological features of C. Apoptosis-Based Shrink and develop bubble-like blebs on cell’s surface apoptotic cells using Therapeutics in Clinical Chromatin (DNA and protein) in the nucleus is Microscopy Trials systematically degraded by nucleases C. DNA Fragmentation Apoptosis involves breakdown of mitochondria with the D. Cell Membrane release of Cytochrome C Alterations Phospholipid phosphatidylserine (PS) is translocated to III. Mechanisms of Apoptosis A. Intrinsic Pathway the outer membrane bilayer (Fig. 2) B. Extrinsic Pathway ○ Initially present in the membrane bilayer intracellularly C. Caspase Cascade (inner layer) ○ Enzyme flippase facilitates flipping of PS ABBREVIATIONS ○ Can be used to signal the neighboring cells that cell PS Phosphatidylserine death has been initiated AIF Apoptosis inducing factor HMW High molecular weight LMW Low molecular weight TUNEL TdT-mediated dUTP nick end labeling TdT Terminal deoxynucleotidyl transferase dUTP Deoxynucleotide triphosphate FITC Fluorescein isothiocyanate LEARNING OBJECTIVES Characterize types of cell death Figure 2. PS translocation from the inner membrane side of the lipid bilayer to Learn the techniques in studying cell death the outer membrane side. Describe the mechanisms of apoptosis Correlate cell death with diseases Apoptosis as a Normal Physiological Process Part of proper development I. CELL DEATH ○ Resorption of tadpole tails A. Necrosis ○ Formation of human fingers and toes Cell death occurs due to mechanical damage (ex. exposure Membranes in between are eliminated via apoptosis to toxic chemicals) or any other environmental factors ○ Proper networking of neurons Characteristic features: Eliminate potentially damaging cells ○ Cell and organelle swelling ○ Cells infected with the viruses ○ Cell content leakage Prevents proliferation of the virus ○ Inflammation of surrounding tissues ○ Cells of the immune system “Messy” death (Fig. 1R) ○ Cells with irreparable DNA damage ○ Cells rupture → cytoplasmic contents get scattered in ○ Cancer cells the neighboring tissues → immune response triggered (ex. inflammation) Table 1. Examples of apoptosis in development ORGAN APOPTOTIC CELLS Lens cells ○ Forms during embryonic development ○ Consists of apoptotic cells that have replaced their innards with the clear protein crystallin Figure 1. Cell Death Photomicrograph. (Left) shows cell death via apoptosis Eye and (Right) shows cell death via necrosis. B. Apoptosis From a Greek word that means “dropping-off” or “falling-off” Induced or programmed cell death Figure 3. Close-Up View of Lens Cell Apoptosis forming the ○ Involves activating a pathway to undergo death lens of the eye Page 1 of 9 | TH: DE GUZMAN, B. | BSR TG 2 | CIOCON, DANAO, DE GUZMAN, K.  BSR 2.04 Cell Death and Degeneration Cells of the Villi membrane Budding (off) Lysis (ruptures) ○ Villi - small finger-like projections of the changes changes in intestinal wall phosphatidylserine Intestines ○ Cells arise at the base of the villi ➜ over several (flipped) distribution days, travel to the tip ➜ eventually die and are Mitochondrial Swelling (also - sloughed off to be replaced by new cells changes ruptures) Skin cells Active changes in ○ Begin life in the deepest layers ➜ migrate to the gene expression Metabolic/ Skin surface in layers, undergoing apoptosis along the (e.g. Bcl2, Bax) Synthetic - way ➜ the resulting dead cell layer forms the Active protein changes protective outer skin layer, called the epidermis synthesis T Lymphocytes (or T-cells) Protease activation ○ White blood cells that are critical components of the immune system II. TECHNIQUES IN STUDYING APOPTOSIS ○ Mature in the thymus gland (located in the C. Apoptotic Parameters upper chest area just below the neck) Table 3. Timeline of apoptotic parameters ○ T-cells that would be ineffective or that would APOPTOTIC TOOLS FOR APOPTOSIS YEAR attack the body's own tissues commit suicide PARAMETER DETECTION Thymus before they have the chance to enter the Membrane blebbing, but no loss bloodstream Morphological of integrity Clinical significance: If T-cells have been found 1972 features of apoptotic Shrinking of cytoplasm to recognize self-antigens, they undergo cells Condensation of nucleus apoptosis to prevent autoimmune disease Formation of apoptotic bodies DNA strand breaks Cells of the uterine wall 1987 DNA fragmentation Apoptotic DNA fragments Uterus ○ Die and are sloughed off during menstruation (DNA-Laddering) Cells that become infected by a virus or that Activation of 1995 Caspase cascade sustain irreparable genetic mutations often proteases commit suicide Cell membrane 1997 Phosphatidylserine translocation alterations Other Clinical significance: Failure of a genetically Alteration of mitochondrial altered cell to commit suicide can contribute to Mitochondrial permeability the development of cancer 1998 changes Release of Cytochrome C and AIF (Apoptosis inducing factor) A. Apoptosis VS Necrosis D. Morphological features of apoptotic cells using Microscopy The type of cell death can be discriminated, whether Microscopy is the most basic tool apoptosis or necrosis, by looking at: With a microscope, the morphology of cells undergoing ○ Cell morphology apoptosis can be visualized ○ Cell DNA ○ Cells with membrane blebbing signify that they are ○ Expression of certain genes undergoing apoptosis Present in apoptosis Absent in necrosis Table 2. Summary of differences between apoptosis and necrosis APOPTOSIS NECROSIS Normal physiological process Anoxia Causes Lack of growth Physical Damage factors Chemical Damage Figure 4. Apoptotic Insect cells (Left) and Mouse cells (Right) with membrane Hormonal influence blebbing under Transmission Electron Microscope (TEM) Mild toxic influence First apparent Shrinking Swelling cellular Convolution changes Condensation Segmentation Nuclear DNA fragmentation - changes ○ “Ladder-like” when visualized on a gel electrophoresis Figure 5. Apoptotic cells with membrane blebbing Cell Surface protrusions Smoothing Page 2 of 9 | TH: DE GUZMAN, B. | BSR TG 2 | CIOCON, DANAO, DE GUZMAN, K.  BSR 2.04 Cell Death and Degeneration DAPI Staining in Fluorescence Microscopy Table 4. DNA Fragmentation facilitated by physical injury vs. enzymes With the advent of fluorescent labels, fluorescence BY ENZYMES BY PHYSICAL INJURY microscopy facilitated targeted visualization of cell (APOPTOSIS) (NECROSIS) structures DNA NOT random Random Useful for the assessment of apoptosis via nuclear Fragmentation fragmentation or nuclear condensation Specific, clear sizes DAPI stain where the nucleases cut Size of DNA NO specific sizes ○ Binds to DNA the DNA and fragmented ○ Stains nuclear material or the nucleus them ○ When the nucleus condenses (sign of apoptosis): DNA Ladder Present Absent Stain intensity = increases Nucleus size = shrinks Distribution of DNA in Normal and Apoptotic cells In a normal cell, the nucleus is intact: ○ High molecular weight (HMW) DNA is present (+) ○ Low molecular weight (LMW) DNA is absent (-) because the nuclear DNA is big In an apoptotic cell, due to the activation of DNA endonucleases: ○ HMW DNA in the nucleus decreases ○ LMW DNA in the nucleus increases ○ LMW DNA in the cytoplasm increases in particular because the contents of the nucleus would be distributed among the apoptotic bodies Table 5. Distribution of DNA in Normal and Apoptotic Cells Figure 6. Stain intensity is greater yet nucleus size is smaller in apoptotic cells with condensed nuclei (red arrows) than the normal cells (white circle). DAPI COMPART NORMAL CELL APOPTOTIC CELL staining of HeLa Cells under fluorescence microscopy. MENT HMW LMW HMW LMW Nucleus + - ⬇ ⬆ Cytoplasm - - - ⬆ Legend: (+) = present/intact; (-) = absent DNA Ladder by Agarose Gel Electrophoresis Figure 7. Apoptotic cell with membrane blebbing (red pointer). DAPI staining of U937 cells under fluorescence microscopy. E. DNA Fragmentation Another approach to detect apoptosis is by analyzing the genomic DNA content of cells DNA ladder appearing on an agarose gel ○ Indicates apoptosis and that nucleases were activated to generate the cut Figure 9. Apoptotic DNA ladder kit. lane 2 = untreated; lane 3 = treated. Table 6. DNA ladder appearance on an agarose gel INTACT NUCLEAR DNA APOPTOTIC DNA Shown on Lane 3 Appearance Shown on Lane 2 DNA appears like a (Fig. 9) NO “ladder” “ladder” DNA is undergoing Nucleus is intact, so it nuclear fragmentation, so Reason only has HMW DNA it has both HMW and Figure 8. Nucleosomal Banding Pattern LMW DNA Page 3 of 9 | TH: DE GUZMAN, B. | BSR TG 2 | CIOCON, DANAO, DE GUZMAN, K.  BSR 2.04 Cell Death and Degeneration HMW DNA can be seen, 2. Add TdT and *FITC-dUTP to cells but the number of cells Other - that are still viable, and *Fluorophore: FITC (Fluorescein isothiocyanate) Information how much underwent apoptosis is not known 3. FITC-dUTP incorporated into DNA In Situ Apoptosis Detection by TUNEL Method TdT-mediated dUTP nick end labeling (TUNEL) Method ○ A newer method, done while cells are still in culture ○ “TdT-mediated dUTP” - labeled molecules 4a. Fluorescence microscopy; OR TdT: terminal deoxynucleotidyl transferase dUTP: deoxynucleotide triphosphate ○ “Nick end labeling” - tagging ends of DNA that have been cut 4b. Light microscopy Labeling principle: the 3’ ends of a DNA strand is extended POD or AP by an X-dNTP that is tagged by a fluorescent molecule Non-specific Staining vs Specific Staining Non-specific staining ○ Cannot discriminate between apoptotic and non-apoptotic cells Specific staining ○ With the TUNEL assay, given the same monolayer, apoptotic cells can easily be identified and localized ○ In contrast to gel electrophoresis method, the TUNEL method can be used to quantify the number of apoptotic cells via the apoptotic index ○ Apoptotic index is determined by: Counting the total number of TUNEL stained fluorescent cells versus the total number of cells ○ Counting can be performed: Figure 10. In situ nick translation (Left) and In situ end labeling (Right) Manually With the use of image analysis software like ImageJ TUNEL Mechanism In a non-apoptotic cell: ○ There are only a few 3’ ends available for labeling Generates a very weak signal Not visible under the microscope In an apoptotic cell: ○ Endonucleases are activated that cut DNA into smaller fragments ○ The more DNA fragments, the more free 3’ ends that are labeled Visible under the microscope Density can measured Hence, in the TUNEL assay, apoptotic cells would give a fluorescent signal that can be visualized under a fluorescence microscope Figure 12. Monolayer of cells with Non-specific staining (Left) where blebbing can be seen; and Specific staining by TUNEL Assay (Right) where fluorescent signals of apoptotic cells can be seen F. Cell Membrane Alterations Flow Cytometry A more complicated but elegant method Recall: when a cell undergoes apoptosis, the Figure 11. Non-apoptotic cell (Left); Apoptotic cell (Right) showing more phosphatidylserine (PS) shifts from the inner membrane DNA fragments and fluorescent signals (in green) leaflet to the outer membrane leaflet ○ Once PS goes into the outer membrane leaflet, an Table 7. TUNEL Staining by in situ Cell Death Detection Kit-Test Principle antibody that binds to PS can be used and it can be DIAGRAM STEPS tagged by a fluorescent probe, then that can be detected using a flow cytometer 1. Cells on slide Given a cell population, a cell mixture in solution, flow cytometer can easily quantify how many cells are undergoing apoptosis and how many are not ○ Very useful technique Page 4 of 9 | TH: DE GUZMAN, B. | BSR TG 2 | CIOCON, DANAO, DE GUZMAN, K.  BSR 2.04 Cell Death and Degeneration A. Intrinsic Pathway Clinical significance: For people who are trying to find potential Activated by signals arising within the cell, i.e. cell stress or anti-cancer drugs, they would want the drug candidates to be DNA damage. selective in triggering cell death. To do that, when they expose the cells to the drug candidate with the extract from a particular plant, it should trigger apoptosis in the cancer cell but should not affect the normal cell. If they are able to show that, then they would have a potential drug candidate assay, chemotherapeutic agent. III. MECHANISMS OF APOPTOSIS What makes a cell decide to end its life (cell death)? ○ Withdrawal of positive signals E.g. intrinsic factors, cell adhesion Positive signals are needed for continued survival, important to keep them viable Figure 14. Intrinsic pathway of apoptosis from DNA replication ○ Receipt of negative signals E.g. death activators, radiation, viral infection, One of the most important intrinsic pathways is brought by cytotoxic T-cells DNA replication. ○ Lack of any signal (or cell isolation) ○ The lagging strand shortens for every DNA replication If cells are too far from adjacent cells, they would not that occurs during cell division. receive signals from the other cells ○ The telomeres found at the end of the chromosomes Thus, the cells in culture must be seeded with a shorten. defined minimum seeding density (number of cells Telomeres protect the actual coding regions. to be placed on the dish for viable growth). When a nucleus undergoes replication, it does not ↪ They are usually kept at 10,000 cells per cm2 matter if the telomeres shorten as long as the actual genes are protected. However, there’s a limit known as the Hayflick limit. Here, continuous cell division will cause the telomeres to get too short which may cause DNA damage and mutations. When the Hayflick limit is reached, this would cause the activation of apoptosis. Figure 15. Intrinsic pathway of apoptosis from the release of cytochrome c. Figure 13. Cells receive many types of signals which can trigger different Another activation of the intrinsic pathway involves the types of responses (survive, divide, differentiate) or if there is no signal, they release of Cytochrome C from the mitochondria. will die ○ Stress factors like oxygen radicals can cause its release. As illustrated in Figure 15, Cytochrome C will be received by Cells can receive many types of signals which can trigger an adaptor protein (Apaf-1). This binding is crucial for the different types of responses: activation of caspases. ○ Given the correct signals the cell will survive (blue) ○ Caspases are the initiators and effectors of apoptosis. ○ Addition of additional signals will trigger the cells to Apaf-1 and Cytochrome C combine with other complexes to proliferate (red). form apoptosomes. ○ Addition of signals may also cause them to differentiate ○ Apoptosomes start to initiate the activation of (green) from a progenitor cell to a differentiated cell. procaspases which would eventually proceed with the ○ However, if you withdraw all the signals coming from caspase cascade. other cells, this would activate the apoptotic cell ○ This caspase cascade would finally trigger the effects of pathway. apoptosis. *TG Note: Some readings were uploaded on NEOLMS regarding the structure of apoptosome, its assembly process, and new insights into how apoptosome regulates apoptosis. Page 5 of 9 | TH: DE GUZMAN, B. | BSR TG 2 | CIOCON, DANAO, DE GUZMAN, K.  BSR 2.04 Cell Death and Degeneration Extrinsic signals from the outside include survival factors, B. Extrinsic Pathway chemokines, growth factors, death activators, chemokines, Triggered when death activators bind to receptors at the etc. cell surface. Intrinsic signals which may come from the mitochondria ○ Examples of death activators: Fas ligand, TNF-𝛼 include caspase 9 and cytochrome C. (cytokine), and lymphotoxin. Can also be triggered by dangerous reactive oxygen species. ○ To prevent the spread of free radicals, a cell would also undergo programmed cell death. Figure 16. Extrinsic pathway of apoptosis from the release of Cytochrome C. Figure 19. Pathways for DNA Damage An external signal may be from a killer lymphocyte which In the intrinsic mechanism, when the DNA damage has been carries the Fas ligand that comes in contact with the Fas observed and the cause is not related to DNA replication protein. (ex. cell exposure to radiation), apoptosis will not always The binding of Fas ligand to Fas protein triggers the happen. activation of procaspase-8, which eventually leads to the ○ Tumor suppressor genes (p53) would initially halt the initiation of the caspase cascade. cell cycle to allow DNA repair before restarting the cell cycle again. ○ However, if the damage that occurred can no longer be repaired, then apoptosis and cell death will be triggered. When equilibrium is sustained, cellular and genetic stability can be achieved even in the presence of cells undergoing apoptosis. C. Caspases - Cysteine Proteases Figure 17. Extrinsic pathway of apoptosis from TNF-a. Another signal that activates the extrinsic pathway come from death activators like TNF-𝛼. These death activators fall and intersect with the caspase activation. Figure 20. Caspase cascade Caspases induce apoptosis in the cell Target site: cleave after Asp residues. There are two main classes of caspases: ○ Initiator caspase Ex. Caspase-9 Upstream activators of the effector caspases ○ Effector caspase Ex. Caspase-3 Executioners in the cell They cleave the proteins that actually induce apoptosis in the cell. Figure 18. Extrinsic pathway and Intrinsic pathway (combined) mechanisms for apoptosis. Page 6 of 9 | TH: DE GUZMAN, B. | BSR TG 2 | CIOCON, DANAO, DE GUZMAN, K.  BSR 2.04 Cell Death an