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  1. 1. APOPTOSIS Dr.Dinesh T Junior resident Department of Physiology JIPMER sclero dinesh
  2. 2. Discussion headings <ul><li>Introduction </li></ul><ul><li>Etiopathogenesis </li></ul><ul><li>Morphological, Biochemical changes </li></ul><ul><li>Mechanism – Intrinsic & Extrinsic pathway </li></ul><ul><li>Disorders of apoptosis </li></ul><ul><li>Conclusion </li></ul>sclero dinesh
  3. 3. Introduction sclero dinesh
  4. 4. Apoptosis - Definition <ul><li>A pathway of cell death induced by a tightly regulated suicidal program, in which the cells destined to die activate enzymes that degrade cells own nuclear DNA and nuclear, cytoplasmic proteins. </li></ul>sclero dinesh
  5. 5. Kerr Wyllie and Currie paper, British Journal of Cancer, 1972 Aug;26(4):239-57 &quot;We are most grateful to Professor James Cormack of the Department of Greek, University of Aberdeen, for suggesting this term. The word &quot;apoptosis&quot; (ἁπόπτωσισ) is used in Greek to describe the &quot;dropping off&quot; or &quot;falling off&quot; of petals from flowers, or leaves from trees. To show the derivation clearly, we propose that the stress should be on the penultimate syllable, the second half of the word being pronounced like &quot;ptosis&quot; (with the &quot;p&quot; silent), which comes from the same root &quot;to fall&quot;, and is already used to describe the drooping of the upper eyelid sclero dinesh
  6. 6. Historical aspects <ul><li>German scientist Carl Vogt - Principle of apoptosis (1842). </li></ul>sclero dinesh
  7. 7. <ul><li>Walther Flemming – Process of programmed cell death (1845). </li></ul>sclero dinesh
  8. 8. <ul><li>John Foxton Ross Kerr – Distinguish apoptosis from traumatic cell death (1962). </li></ul>sclero dinesh
  9. 9. <ul><li>Nobel prize in 2002 – Sydney Brenner , Horvitz, </li></ul><ul><li>John Buston. </li></ul>sclero dinesh
  10. 10. Cell death mechanisms Death by suicide Death by injury sclero dinesh
  11. 11. sclero dinesh APOPTOSIS NECROSIS NATURAL YES NO EFFECTS BENEFICIAL DETRIMENTAL Physiological or pathological Always pathological Single cells Sheets of cells Energy dependent Energy independent Cell shrinkage Cell swelling Membrane integrity maintained Membrane integrity lost
  12. 12. sclero dinesh APOPTOSIS NECROSIS Role for mitochondria and cytochrome C No role for mitochondria No leak of lysosomal enzymes Leak of lysosomal enzymes Characteristic nuclear changes Nuclei lost Apoptotic bodies form Do not form DNA cleavage No DNA cleavage Activation of specific proteases No activation Regulatable process Not regulated Evolutionarily conserved Not conserved Dead cells ingested by neighboring cells Dead cells ingested by neutrophils and macrophages
  13. 13. Significance of apoptosis <ul><li>During development many cells are produced in excess which eventually undergo programmed cell death and thereby contribute to sculpturing many organs and tissues [ Meier, 2000 ] </li></ul><ul><li>In human body about one lakh cells are produced every second by mitosis and a similar number die by apoptosis ( Vaux and Korsmayer ,1999, cell ) </li></ul><ul><ul><li>Between 50 and 70 billion cells die each day due to apoptosis in the average human adult. For an average child between the ages of 8 and 14, approximately 20 billion to 30 billion cells die a day. ( Karam, Jose A. (2009). Apoptosis in Carcinogenesis and Chemotherapy. Netherlands: Springer. ISBN 978-1-4020-9597-9 ) </li></ul></ul>sclero dinesh
  14. 14. Etiopathogenesis sclero dinesh
  15. 15. Why should a cell commit suicide? <ul><li>1. Programmed cell death is as needed for proper normal development as mitosis is. </li></ul><ul><ul><li>Examples: </li></ul></ul><ul><ul><ul><li>The resorption of the tadpole tail in frog . </li></ul></ul></ul><ul><ul><ul><li>The formation of the fingers and toes of the fetus requires the removal, by apoptosis. </li></ul></ul></ul><ul><ul><ul><li>The sloughing off of the endometrium at the start of menstruation. </li></ul></ul></ul><ul><ul><ul><li>The formation of the proper connections (synapses) between neurons in the brain. </li></ul></ul></ul>sclero dinesh
  16. 16. <ul><li>2. Programmed cell death is needed to destroy cells that represent a threat to the integrity of the organism. </li></ul><ul><li>Examples: </li></ul><ul><ul><li>Cells infected with viruses </li></ul></ul><ul><ul><li>Cells of the immune system </li></ul></ul><ul><ul><li>Cells with DNA damage </li></ul></ul><ul><ul><li>Cancer cells (Uncontrolled proliferated cells) </li></ul></ul>sclero dinesh
  17. 17. <ul><li>Apoptosis in physiologic situations </li></ul><ul><li>Programmed destruction during embryogenesis </li></ul><ul><li>Involution of hormone dependent tissues </li></ul><ul><li>Cell loss in proliferating cell populations </li></ul><ul><li>Elimination of harmful self- reactive lymphocytes </li></ul><ul><li>Death of host cells </li></ul>sclero dinesh
  18. 18. <ul><li>Apoptosis in bud formation during which many interdigital cells die.  They are stained black by a TUNEL method </li></ul><ul><li>   </li></ul><ul><li>Incomplete differentiation in two toes due to lack of apoptosis </li></ul>sclero dinesh
  19. 19. Apoptosis: in embryogenesis Morphogenesis (eliminates excess cells): Selection (eliminates non-functional cells): sclero dinesh
  20. 20. Apoptosis: in embryogenesis Immunity (eliminates dangerous cells): Self antigen recognizing cell Organ size (eliminates excess cells): sclero dinesh
  21. 21. Apoptosis: importantance in adults Tissue remodeling (eliminates cells no longer needed): Virgin mammary gland Late pregnancy, lactation Involution (non-pregnant, non-lactating) Apoptosis Apoptosis - Testosterone Prostate gland sclero dinesh
  22. 22. Apoptosis: importantance in adults Tissue remodeling (eliminates cells no longer needed): Resting lymphocytes + antigen (e.g. infection) - antigen (e.g. recovery) Apoptosis sclero dinesh
  23. 23. <ul><li>Apoptosis in pathological conditions </li></ul><ul><li>- DNA damage </li></ul><ul><li>- Accumulation of misfolded proteins </li></ul><ul><li>- Cell death in certain infections </li></ul><ul><li>- Pathological atrophy in parenchymal organs </li></ul>sclero dinesh
  24. 24. <ul><ul><li>Cells of the immune system </li></ul></ul><ul><ul><ul><li>CTLs induce apoptosis in each other and even in themselves. </li></ul></ul></ul><ul><ul><ul><li>Defects in the apoptotic machinery is associated with autoimmune diseases such as lupus erythematosus and rheumatoid arthritis. </li></ul></ul></ul>sclero dinesh
  25. 25. <ul><ul><li>Cells infected with viruses </li></ul></ul><ul><ul><ul><li>One of the methods by which cytotoxic T lymphocytes (CTLs) kill virus-infected cells is by inducing apoptosis </li></ul></ul></ul>sclero dinesh
  26. 26. <ul><ul><li>Cells with DNA damage </li></ul></ul><ul><ul><ul><li>Damage to its genome can cause a cell </li></ul></ul></ul><ul><ul><ul><ul><ul><li>to disrupt proper embryonic development leading to birth defects </li></ul></ul></ul></ul></ul><ul><ul><ul><ul><ul><li>to become cancerous. </li></ul></ul></ul></ul></ul><ul><ul><ul><li>Cells respond to DNA damage by increasing their production of p53 . p53 is a potent inducer of apoptosis. </li></ul></ul></ul>sclero dinesh
  27. 27. <ul><ul><li>Cancer cells </li></ul></ul><ul><ul><ul><li>Radiation and chemicals used in cancer therapy induce apoptosis in some types of cancer cells. </li></ul></ul></ul>Fig. 1:  SC-1 induced apoptosis in stomach carcinoma cells         Left: Before induction        Middle: 24h after induction         Right: 48h after induction sclero dinesh
  28. 28. Morphological & Biochemical changes sclero dinesh
  29. 29. Classic changes <ul><li>Cell shrinkage </li></ul><ul><li>Nuclear fragmentation </li></ul><ul><li>Chromatin condensation </li></ul><ul><li>Chromosomal DNA fragmentation </li></ul><ul><li>Formation of cytoplasmic blebs& apoptotic bodies </li></ul><ul><li>Phagocytosis </li></ul>sclero dinesh
  30. 30. Histology <ul><li>Apoptotic bodies </li></ul><ul><li>Round oval mass of intensely eosinophillic cytoplasm </li></ul><ul><li>Fragments of dense nuclear chromatin </li></ul>sclero dinesh
  31. 31. Bio chemical changes sclero dinesh
  32. 32. Mechanisms of apoptosis sclero dinesh
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  35. 35. Caspases <ul><li>Caspase are Cysteine- Aspartic acid specific proteases that mediates the events that are associated with programmed cell death. </li></ul><ul><li>Their catalytical activity depends on a critical cysteine-residue within a highly conserved active-site pentapeptide QACRG, </li></ul><ul><li>Caspases specifically cleave their substrates after Asp residues. </li></ul><ul><li>Caspase- 8, Caspase- 9 </li></ul><ul><ul><ul><li>acts as an initiator of the caspase activation cascade . </li></ul></ul></ul><ul><li>Caspase-3 </li></ul><ul><ul><ul><li>key effector in the apoptosis pathway, amplifying the signal from initiator caspases and signifying full commitment to cellular disassembly. </li></ul></ul></ul>sclero dinesh
  36. 36. Initiation <ul><li>Absence of stimuli - hormones, growth factors </li></ul><ul><li>Activation of receptors – TNF family </li></ul><ul><li>Heat ,radiation, chemicals </li></ul><ul><li>Genetically programmed events </li></ul>sclero dinesh
  37. 37. STAGES OF CLASSIC APOPTOSIS Healthy cell DEATH SIGNAL / STIMULI (extrinsic or intrinsic) Commitment to die (reversible) EXECUTION (irreversible) Dead cell (condensed, crosslinked) ENGULFMENT (macrophages, neighboring cells) DEGRADATION sclero dinesh
  38. 38. Receptor pathway (physiological): Death receptors: (FAS, TNF-R, etc) FAS ligand TNF Death domains Adaptor proteins Pro-caspase 8 (inactive) Caspase 8 (active) Pro-execution caspase (inactive) Execution caspase (active) Death MITOCHONDRIA sclero dinesh
  39. 39. Apoptosis triggered by external signals: the extrinsic or death receptor pathway sclero dinesh
  40. 40. Regulation of apoptosis <ul><li>Regulatory proteins – BCL -2, equivalent to CED -9 </li></ul><ul><li>Apoptosis depends on binding of BCL -2 with pro apoptotic and anti apoptotic proteins. </li></ul><ul><li>Situated in the outer mitochondrial membrane. </li></ul><ul><li>Apaf -1 equivalent to CED -4. </li></ul><ul><li>Tp 53, caspases, BAX, viruses such as adeno, papilloma , hepatitis B. </li></ul>sclero dinesh
  41. 41. Intrinsic pathway (damage): Mitochondria Cytochrome c release Pro-caspase 9 cleavage Pro-execution caspase (3) cleavage Caspase (3) cleavage of cellular proteins, nuclease activation, etc. Death BAX BAK BOK BCL-Xs BAD BID B IK BIM NIP3 BNIP3 BCL-2 BCL-XL BCL-W MCL1 BFL1 DIVA NR-13 Several viral proteins sclero dinesh
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  44. 44. Intracellular signals Oxidative damage from free radicals, Radiation, Virus infection, Nutrient deprivation, Pro-apoptotic Factors Damage to the mitochondrial membrane increasing permeability Entry of Cytochrome C into the cytoplasm Cytochrome C binds to Apaf-1 forming an apoptosome Apoptosome activates procaspase-9 to caspase-9 Caspase-9 cleaves and activates caspase-3 and caspase-7. This executioner caspases activate a cascade of proteolytic activity that leads to: Chromatin condensation, DNA fragmentation, Protein cleavage, Membrane permeability sclero dinesh
  45. 45. sclero dinesh
  46. 46. Physiological Intrinsic receptor pathway damage pathway MITOCHONDRIAL SIGNALS Caspase cleavage cascade Orderly cleavage of proteins and DNA CROSSLINKING OF CELL CORPSES; ENGULFMENT (no inflammation) sclero dinesh
  47. 47. Steps sclero dinesh
  48. 48. Disorders of apoptosis sclero dinesh
  49. 49. Apoptosis: Role in Disease TOO MUCH: Tissue atrophy TOO LITTLE: Hyperplasia Neurodegeneration Thin skin etc Cancer Athersclerosis etc sclero dinesh
  50. 50. Apoptosis: Role in Disease Neurodegeneration <ul><li>Neurons are post-mitotic (cannot replace themselves; neuronal stem cell </li></ul><ul><li>replacement is inefficient) </li></ul><ul><li>Neuronal death caused by loss of proper connections, loss of proper growth </li></ul><ul><li>factors (e.g. NGF), and/or damage (especially oxidative damage). </li></ul><ul><li>Neuronal dysfunction or damage results in loss of synapses or loss of cell </li></ul><ul><li>bodies (synaptosis, can be reversible; apopsosis, irreversible) </li></ul><ul><li>PARKINSON'S DISEASE </li></ul><ul><li>ALZHEIMER'S DISEASE </li></ul><ul><li>HUNTINGTON'S DISEASE etc. </li></ul>sclero dinesh
  51. 51. Apoptosis: Role in Disease Cancer <ul><li>Apoptosis eliminates damaged cells (damage => mutations => cancer </li></ul><ul><li>Tumor suppressor p53 controls senescence and apoptosis responses to damage. </li></ul><ul><li>Most cancer cells are defective in apoptotic response(damaged, mutant cells survive) </li></ul><ul><li>High levels of anti-apoptotic proteins </li></ul><ul><li>or </li></ul><ul><li>Low levels of pro-apoptotic proteins ===> CANCER </li></ul>sclero dinesh
  52. 52. <ul><li>Apoptosis: Role in Disease </li></ul><ul><li>Cancer </li></ul><ul><li>Virus associated cancer </li></ul><ul><li>Several human papilloma viruses ( HPV ) have been implicated in causing cervical cancer. One of them produces a protein ( E6 ) that binds and inactivates the apoptosis promoter p53 . </li></ul><ul><li>Epstein-Barr Virus ( EBV ), the cause of mononucleosis and associated with some lymphomas </li></ul><ul><ul><li>produces a protein similar to Bcl-2 </li></ul></ul><ul><ul><li>produces another protein that causes the cell to increase its own production of Bcl-2. Both these actions make the cell more resistant to apoptosis (thus enabling a cancer cell to continue to proliferate). </li></ul></ul>sclero dinesh
  53. 53. <ul><li>Some B-cell leukemia and lymphomas express high levels of Bcl-2 , thus blocking apoptotic signals they may receive. The high levels result from a translocation of the BCL-2 gene into an enhancer region for antibody production. </li></ul><ul><li>Melanoma (the most dangerous type of skin cancer) cells avoid apoptosis by inhibiting the expression of the gene encoding Apaf-1 . </li></ul>Apoptosis: Role in Disease Cancer sclero dinesh
  54. 54. <ul><li>Other cancer cells express high levels of FasL , and can kill any cytotoxic T cells (CTL) that try to kill them because CTL also express Fas (but are protected from their own FasL). </li></ul><ul><li>Some cancer cells, especially lung and colon cancer cells, secrete elevated levels of a soluble &quot;decoy&quot; molecule that binds to FasL, plugging it up so it cannot bind Fas. Thus, cytotoxic T cells (CTL) cannot kill the cancer cells </li></ul>Apoptosis: Role in Disease Cancer sclero dinesh
  55. 55. Apoptosis: Role in Disease Aging Aging --> both too much and too little apoptosis (evidence for both) Too much (accumulated oxidative damage?) ---> tissue degeneration Too little (defective sensors, signals? ---> dysfunctional cells accumulate hyperplasia (precancerous lesions) sclero dinesh
  56. 56. Apoptosis: Role in Disease <ul><li>Apoptosis and AIDS </li></ul><ul><ul><li>Hallmark- the decline in the number of the patient's CD4+ T cells (normally about 1000 per microliter (µl) of blood). </li></ul></ul>sclero dinesh
  57. 57. <ul><li>In Immune system </li></ul><ul><li>Very rarely humans are encountered with genetic defects in apoptosis. </li></ul><ul><li>The most common one is a mutation in the gene for Fas </li></ul><ul><li>mutations in the gene for FasL or even one of the caspases are occasionally seen. </li></ul><ul><li>Autoimmune lymphoproliferative syndrome or ALPS. </li></ul>sclero dinesh
  58. 58. Conclusion sclero dinesh
  59. 59. Cells are balanced between life and death DAMAGE Physiological death signals DEATH SIGNAL PROAPOPTOTIC PROTEINS (dozens!) ANTIAPOPTOTIC PROTEINS (dozens!) DEATH sclero dinesh
  60. 60. Thank u…. sclero dinesh