apoptosis in cells

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2. The history of Apoptosis
 What Apoptosis means
 Significance of Apoptosis.
 Morphological changes
 Major players in apoptosis.
 Pathways to apoptosis
 Therapeutic implications.
 Pathological implication
 Detection of apoptotic cells
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3. The History of Apoptosis
John Kerr:
a Brisbane pathologist in 1972
introduced a concept ‘Apoptosis’.
(Richardson, et al., 2000)
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4. Synonyms: - Antonym: -
Programmed cell death. Necrosis
Programmed cellcell death are of two types: -are of two types: -
Apoptosis : -(Type I)
Autophagic : -(Cytoplasmic, or Type II)
The formation of large vacuoles that eat away organelles
before the nucleus is destroyed.
(Offermanns & Rosenthal , 2004)
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5.  Greek origin word known ‘Falling off or Dropping off.’
 Self destruction programmed genetically sequenced in
biomedical events.
 It’s a kind of counterbalance by elimination of similar
number of cells.
 Death by design.
Apoptosis: -
(Rang & Dale, 2008)
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6. Significance of Apoptosis
 Deletion of dangerous & damage cells.
 Development of organs
 Without apoptosis human
gut can grow 19 km,whole
epithelial lining changes every
23 days.
 Regulation of immune system(T-cells)
Incomplete differentiation
due to lack of apoptosis
 Cancer & Neurodegenerative diseases.
(Gewies, 2003)
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7. Apoptosis Necrosis
• Energy dependent PCD • Cell dies due to damage
• One cell • Group of cells
• No inflammation • Acute inflammation
• Role of mitochondria • No role of mitochondria
• Dead cells eaten by
macrophages
• Dead cells removed by
macrophages.
• Cell shrinkage • Cell swelling
(Haiat, 2006)
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8. The Apoptotic Cell
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9. Morphological changes in Apoptosis
(Hengartner, 2000)
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10. Major players in Apoptosis
Apoptosis Initiating factor(AIF):-
Release from mitochondria enter in
nucleus & trigger cell suicide.
Caspases
Bcl-2 Proteins
 Apoptosome
 p53 gene and p53 protein
(Rang & Dale, 2008)
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11. • It is family of cystein protease.
• present in cell in inactive form.
• Cleave protein bearing a amino acid
sequence located at the terminal.
(cystein aspartic acid specific protease)
(Hill,et al., 2003)
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12. Caspases
14 different Caspases14 different Caspases
Initiator
Caspases
Effectors
Caspases
2,8,9,10.2,8,9,10.
3,6,7.3,6,7.
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13. 13

14. Apoptosis carried out by Caspases
(Susin, et al., 1999)
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15. CARD/DED
Domain
Large subunit
Small subunit
Procaspase Structure
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16. Caspase
activation
Inactive
Procaspase
Proteolysis
cleavage
Activation of
Caspase
Initiator Caspase
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17. Active Initiator
Caspase
Cleavage by protein-protein
interaction
Activation of
effector caspase
Caspase
activation 17

18. The Bcl-2 Family
 Regulator Protein of Mitochondria.
 Isolated from gene involve in B-cell lymphoma.(Bcl-2)
 All have BH3 domain (Bcl-2 Homology)
BH3:-It is Proapoptotic domain expose on activation.
(Dash, et al., 2001)
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19. Bcl-2 family members:-
 BH1-4: - Anti-apoptotic proteins
Bcl-2, Bcl-xL .
 BH1-3: - Pro-apoptotic proteins
Bax, Bok, Bak.
 BH 3: - Only pro-apoptotic proteins
Bad, Bik, Bid.
Bcl-2: -ß-cell Lymphoma, BH:-Bcl-2 Homolog, Bid: - Bcl-2 interacting
domain, Bcl-xL :- x=Close homolog & L= inhibit apoptosis.
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20. Functions:-
 Regulate the balance between Pro-Apoptotic &
anti-apoptotic.
 Involved in Intrinsic pathway.
 Induction of Bcl-2 is by death signals.
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21. Apoptosome
 Multisubunit protease activation complex.
 Form after induction of death signal in mitochondrial
pathway.
 Discovery of Apoptosome by ‘prof. Wang’.
 Wheel-like structure connected to seven radial spokes
(Adrain, 2001; Dash, 2003)
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22. Active caspase
Apoptic
stimuli
Apoptic
stimuli
Cytochrome-c
Binding
Apaf-1
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23. Apoptotic activation
protease factor-1
Apoptotic activation
protease factor-1
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24. p53 gene & p53 protein
 p53 is a tumor suppressor gene.
 Prevent completion of cell cycle.
 If DNA damage occurs activation of gene p53 response to
cell death.
 Curable damage can be repaired
 In curable damage of DNA leads to cell death
(Dlamini et al.,2005)
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25. Pathways to Apoptosis
1) Death receptor pathways /(Extrinsic Pathway)
2) Mitochondrial pathways /(Intrinsic Pathway)
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26. Death receptor pathways /(Extrinsic Pathway)
 Induction is mediated through cell surface receptors
know to be ‘Death receptors’.
 Death ligands are activating these receptors.
 Further activation of Caspases leads to cell death
(Rang & Dale, 2008)
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27. Death Receptors
TNFR - Super family
TNFR-Tumor necrosis factor Receptor, CD: -Cluster differentiation
TRAIL- Tumor necrosis factor –α related apoptosis inducing ligand
Fas:- Apoptosis stimulating fragment.
Death receptorDeath receptor Death ligandsDeath ligands
 TNFR-1
 CD95 (Apo-1) (Fas) (TNFR-2)
 TRAIL-R1 (DR-4)
 TRAIL-R2 (DR-5)
TNF-α
CD95L (FasL)
TRAIL-R1
TRAIL-R2
(Ashkenazi, 2002)
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28. TNF Receptor signaling
Binding of Ligand
to Death receptor
Binding of Ligand
to Death receptor
Binding of adapter
protein with
Activation of
Another protein
TRAF-2
Binding of adapter
protein with
Activation of
Another protein
TRAF-2
TRAF-2: -TNF associated factor -2,
TRADD:- TNF associated death domain,
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29. Fas:- Apoptosis stimulating fragment ., FADD:- Fas Associated death domain
Adapter Protein
Fas signaling pathway
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30. Death receptors signal transductionDeath receptors signal transduction
Effectors domain
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31. The Extrinsic Apoptosis Pathway
FADD, Fas-associated death domain, DR:- Death receptors.
Pro-apoptotic ligands
Caspase 3, 6, 7
Apoptosis
FADD
DR5
DR4
Procaspase 8, 10
Caspase 8, 10
Plasma
membrane
(Ashkenazi A. Nat Rev Cancer 2002;2:420–430.)
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32. Macrophage release anti-inflammatory mediators like
[transforming growth factor(TGF-ß] & IL-10]
Macrophage undergo phagocytosis of cell
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33. Mitochondrial pathways /(Intrinsic Pathway)
 Release of protein from mitochondrial inner membrane
into cytosol.
 DNA damage induce apoptosis.
 Cellular stress, heat shock, oxidative stress, cellular
damage, that are also induced apoptosis.
(Martin, et al., 2003)
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34. Plasma
membrane
Cytosol
DNA
Damage
P53 proteinP53 protein
Pro-apoptic Bcl-2
Mitochondria
Apaf-1
Cytochrome-c
Procaspase-9 34

35. Caspase-9Caspase-9
Caspase-3Caspase-3
Activation
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36. APOPTOSIS
Video
(http://www.researchapoptosis.com) 36

37. Apoptosis By Injury
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38. Pathological Implications
 Growth of tissue & organs in embryo.
 Replenishment of time-expire cells like gut-epithelium.
 Repair & healing after injury or inflammation.
 Regeneration of tissue.
 Hyperplasia.
(Barton, et al., 2004)
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39. Therapeutic implications
 Neurodegenerative disease like Alzheimer’s disease.
 Myocardial infraction.
 Targets for anti-cancer drugs.
(Gerald, 2002)
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40. Neurodegenerative disease: -Neurodegenerative disease: -
 Alzheimer’s disease show excessive Apoptosis.
 Neuronal growth factor & brain derived
neurotrophic factors.
 Secrets protein that maintain the balance Pro-
Apoptotic & Anti-Apoptotic.
 Blocking of these factor are new area of interest for
Target drug delivery.
 Alzheimer’s disease show excessive Apoptosis.
 Neuronal growth factor & brain derived
neurotrophic factors.
 Secrets protein that maintain the balance Pro-
Apoptotic & Anti-Apoptotic.
 Blocking of these factor are new area of interest for
Target drug delivery.
(Gerald, 2002)
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41. Apoptotic Neuron
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42. Myocardial Infraction: -Myocardial Infraction: -
 Occur due to blockage of coronary artery by
thrombus.
 Decrease in oxygen supply leads to (M.I.) by necrosis
or Apoptosis
 Two pathways of cell death
1) Necrosis 2) Apoptosis
(Rang & Dale, 2008)
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43. Current approach are finding to
inhibit these pathway which are
beneficial in clinical
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ICE:-interleukin -1 converting enzyme.
PARP:- (poly-ADP-ribose-polymerase .

44. Targets for Anti-cancer drugs: -Targets for Anti-cancer drugs: -
 Bcl-2 family as a target for new drug.
 Bcl-2 is Anti-apoptotic & increase resistance to
cancer chemotherapy.
 Death receptor & there respective ligands are useful
in targeting the Anti-cancer drug.
(Fischer, et al., 2005)
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45. How do we recognize Programmed Cell
Death?
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46. Detection of apoptic cells
 Electron Microscopy
 Fluorescence Microscopy
 Staining with Eosin
 Detection of PCD Based on Morphology
 Detection of DNA Fragmentation
 Agarose Gel Electrophoresis
 DNA Fragments by Cell Fractionation
 Detection of DNA Fragmentation by Filtration Assay.
(Kaufmann, 2000)
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47.  Terminal Deoxyribonucleotidyl Transferase-Mediated
dUTP Nick End Labeling.(TUNEL)
 Flow Cytometry.
 Enzyme Assay.
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48. Detection Based on Morphology: -Detection Based on Morphology: -
 Shrinkage of the cell.
 Detachment from its neighbors.
 Fragmentation of nucleus.
 Packaging of the cell into multiple plasma
membrane.
 Phagocytosis of these fragments.
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49. Control Apoptotic cells
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50. Electron Microscopy: -
 A beam of electron using electric & magnetic field
to form enlargement of image on photographic
plate.
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51. Electron Microscopy: -
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52. Detection of DNA fragmentation by Gel
Electrophoresis.
Detection of DNA fragmentation by Gel
Electrophoresis.
 Agarose gel with suitable separation properties.
 Application of total cellular DNA to an Agarose.
 The fragments are spared throughout agarose gel.
 Fragments will be detected by Ethidium bromide.
(Anders, 2002)
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53. 1. DNA from apoptotic cells.
2. DNA isolated from necrotic
cells
3. No samples
4. DNA from normal cell
5. DNA from normal cell
6. No sample
Gel Electrophoresis
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54. Flow CytometryFlow Cytometry
 Well known as a ‘Flow Microfluriometer.
 To study Normal & Malignant Lymphocytes.
 Measures amount of fluorescence associated with cell.
 Photomultiplier tube is a detector.
 Analyzing the population of cell.
 Around 20,000 cells can be analyzed in few min.
(Kaufmann, 2000)
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http://www.kimmelcancercenter.org
Flow Cytometry

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Advance Flow
Cytometry
Advance Flow
Cytometry
http://www_biotech_missouri_edu

57. Adrain, C. Martin, S.J. 2001 The mitochondrial apoptosome: A killer
unleashed by the cytochrome seas. Trends Biochem. Sci. 26, 390–397.
Ashkenazi, A.Dixit, V. M., 1999. Apoptosis control by death and decoy
receptors. Current Opinion in Cell Biology 11, 255-60.
Dash, P., 2001. Apoptosis Basic Medical Sciences
St.George’s, University of London
Fischer, U.OsthofF, K. S., 2005. New Approaches and Therapeutics
Targeting Apoptosis in Disease: Pharmacological reviews, 57 (2), p.187-
215.
Hill, M.M. Adrain, C. Martin S.J., 2003.Molecular Cell Biology
Laboratory, Department of Genetics Smurfit Institute, 3 (1) p.19-24.
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58. Gewies, A., 2003. Introduction to Apoptosis :Apo Review p.1-26
Hill, M. M.Adrain, C., 2003. Portrait of the killer the mitochondrial
Apoptosome emerges from the shawdo:molecular intervention. 3
(1)p.19-24.
Kaufmann, S.2001. Apoptosis:Phaarmacological implication and
therapeutic opportunites.New York: Academic Press
Li, L.Y., Luo, X. Wang, X., 2001 Endonuclease G is an apoptotic
DNase when released from mitochondria. Nature 412, 95–99.
Margaret, K.T. Cohen, J., 2001. Standard Quantitative Assays for
Apoptosis: Molecular Biotechnology, 19 p.305-315
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59. Offermanns, S. Rosentha, W., 2004. Encyclopedic Reference of
Molecular Pharmacology,Springer
Rang, H. P. Dale, M. M., 2008.Cell proliferation and Apoptosis:
Pharmacology.6th
ed.Churchill Livingstone Elsevier.p.72-88.
http://www.researchapoptosis.com
http://www.apoptosisinfo.org
http:// www.bitessizebio.com
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