Apoptosis basics + microphotographs

1. 1. Apoptosis-101 Presented on- Date- 21-11-14 aj

2. 2. Cell response to stressful conditions and injurious stimuli
When limits of adaptive responses are exceeded or if cells
are exposed to injurious agents or stress, deprived of
essential nutrients, or become compromised by mutations
that affect essential cellular constituents, a sequence of events
follows that is termed cell injury Cell death, the end result
of progressive cell injury two principal pathways of cell
death, necrosis apoptosis aj
3. 3. Apoptosis (Greek origin)=falling off 1842- Vogt gave
first principles of apoptosis 1885- Flemming gave more
precise description 1965- John Kerr using EM
distinguished it from traumatic cell death 1972- coining of
term apoptosis by Kerr, Wyllie, Currie 1990 onwards- use
of newer methods to study proteins and genetics has given
newer insights into apoptosis. aj
4. 4. Definition Apoptosis is a pathway of cell death that is
induced by an internally regulated program in which cells
destined to die activate intrinsic enzymes that degrade the
cells own nuclear DNA and also nuclear and cytoplasmic
proteins With minimal host reaction. aj
5. 5. Causes Physiological Pathological Death by apoptosis is a
normal phenomenon that serves to eliminate cells that are no
longer needed, and to maintain a steady number of various
cell populations in tissues. Apoptosis eliminates cells that are
injured beyond repair without eliciting a host reaction, thus
limiting collateral tissue damage. aj
6. 6. Apoptosis in physiological conditions Destruction of cells
during embryogenesis Implantation Organogenesis
Involution of hormone-dependent tissues upon hormone
withdrawal Endometrial cell breakdown ovarian follicular
atresia in menopause, Cell loss in proliferating cell
populations Immature lymphocytes in the bone marrow and
thymus and B lymphocytes in germinal centers that fail to
express useful antigen receptors aj

7. 7. Apoptosis in pathological conditions DNA damage

Radiation, cytotoxic anticancer drugs, and hypoxia can
damage DNA. If repair mechanisms cannot cope with the
injury, the cell triggers intrinsic mechanisms that induce
apoptosis. In these situations elimination of the cell may be a
better alternative than risking mutations in the damaged
DNA, which may result in malignant transformation.
Accumulation of misfolded proteins ER stress, which
culminates in apoptotic cell death. Apoptosis caused by the
accumulation of misfolded proteins has been invoked as the
basis of several degenerative diseases of the central nervous
system and other organs. Cell death in certain infections,
particularly viral infections loss of infected cells is largely
due to apoptosis that may be induced by the virus or by the
host immune response (as in viral hepatitis). Pathologic
atrophy in parenchymal organs after duct obstruction
Pancreas, parotid gland, and Kidney aj
8. 8. Morphological changes aj
9. 9. Morphological changes characterizing apoptosis Cell
shrinkage Chromatin condensation Formation of
cytoplasmic blebs and apoptotic bodies Phagocytosis of
apoptotic cells aj
10.10. Cell shrinkage Cell becomes smaller in size
Cytoplasm is dense Organelles tightly packed aj
11.11. Chromatin condensation Chromatin aggregates
peripherally under nuclear membrane in form of irregular
dense masses (pyknosis) The nucleus may break up into
fragments (karyorrhexis) aj
12.12. Cytoplasmic blebs and apoptotic bodies Apoptotic cells
first undergoes surface blebbing Then it fragments into
many membrane bound apoptotic bodies (karyolysis) aj
13.
13. Phagocytosis of apoptotic cells Phagocytes ingest
apoptotic cells and degradation occurs by lysosomal
enzymes. Phagocytic cells- macrophages aj

14.14. A) Low power --cell shrinkage in apoptotic cell B)

High power --apoptotic cell nucleus showing chromatin
condensation In both surrounding cells are normal A aj
15.
15. aj
16.
16. Mechanism of apoptosis aj
17.
17. Apoptosis mechanism aj
18.18. Apoptosis mechanism Apoptosis results from the
activation of enzymes called caspases (which exist as inactive
proenzymes, or zymogens). Phases of apoptosis-
Initiation- phase of activation of caspases Executionactivated caspases trigger cell degradation. aj
19.
19. Two Pathways of initiation of apoptosis 1. Intrinsic
2. Extrinsic Mitochondrial Death-receptor initiated
Initiator phase aj
20.20. An overview of the extrinsic and intrinsic pathways.
Both pathways lead to activation of caspase-3 and give rise to
apoptotic cell death. aj
21.21. Intrinsic pathway Major mechanism of apoptosis in all
mammalian cells. Increased permeability of the
mitochondrial outer membrane with consequent release of
death-inducing (pro-apoptotic) molecules from the
mitochondrial intermembrane space into the cytoplasm.
The release of mitochondrial pro-apoptotic proteins is tightly
controlled by the BCL2 family of proteins aj
22.22. BCL2 family of proteins BCL2 stands for B-cell
lymphoma 2. aj
23.23. BCL2 family of proteins Group Members Details Antiapoptotic BCL2, BCL-XL MCL1 Present in outer
mitochondrial membrane Prevent leakage of cytochrome-c
and other death inducing proteins into cytosol. Pro-apoptotic
BAX BAK On activation they increase mitochondrial
membrane permeability. Sensors BAD BIM, BID Puma,
Noxa Act as sensors of cellular stress and damage, and
regulate the balance between the other two groups. aj
24.24. When cells are deprived of survival signals DNA is
damaged misfolded proteins Sensor proteins activate and

further activate pro- apoptotic proteins. Also anti-apoptotic

factor production decreases. With this several
mitochondrial proteins (mainly cyt-c) is released into cytosol.
aj
25.25. Cyt-c in cytosol Cytochrome c binds to a protein called
APAF-1 (apoptosis-activating factor-1), which with effect of
ATP forms the apoptosome. Apoptosome activates caspase
activation cascade and leads to execution phase of apoptosis.
aj
26.26. SMAC proteins in cytosol Mitochondrial proteins
known as SMACs (small mitochondria-derived activator of
caspases) are released into the cytosol following an increase
in permeability. SMACs neutralize inhibitor of apoptosis
proteins (IAPs). This leads to caspase activation thus
apoptosis proceeds to execution phase. aj
27.
27. Extrinsic pathways This pathway is initiated by
engagement of plasma membrane death receptors on a
variety of cells. These death receptors belong to TNF
receptor family. These receptors link with a external
protein to form a complex termed death domain which is
important to activate apoptosis. Important death
receptor activating proteins are TNF Fas ligand
aj
28.28. TNF pathway TNF is Produced by macrophages
Main external signal of apoptosis TNF links with TNFR1 to
form death domain termed TRADD TRADD activates
caspase 8 which initiates apoptosis. aj
29.
29. Fas pathway Fas ligand(FasL) is present on NK
cells Other T cells 3Fas and FasL bind to form Fasassociated death domain (FADD) This activates procaspase 8 to an active form. Caspase 8 initiates the
further execution phase. aj
30.30. Execution phase Both initiating pathways converge at
level of caspase activation. Initiator caspases(8,9,10)
activate the executioner caspases(3,6). These executioner
caspases signal DNA cleavage. DNA clevage causes

disintegration of nucleus(karyorrhexis) and also damages

nuclear membrane. aj
31.
31. Clearing of dead cells Clearing of apoptotic cells
is the final step of apoptosis and in healthy individuals this
process is efficient and rapid enough to prevent any
inflammation and necrosis. Phagocytic cellsmacrophages. aj
32.32. Signals= substances expressed on cell surface Finding
of apoptotic cells- find me signals fractalkine, LPC, S1P
Start of eating of apoptotic cells-eat me signals
phosphatidylserine flip out Expression of C1q,
thrombospondin Viable cells giving out-dont eat me
signals CD47 expression aj
33.
33. aj
34.34. Standard H&E-stained section of lymph node showing
macrophages (arrows) of a germinal centre containing
engulfed remnants of apoptotic cells.aj
35.
35. aj
36.
36. Few examples of apoptosis- aj
37.37. In this fetal thymus there is involution of thymic
lymphocytes by the mechanism of apoptosis. Individual
cells fragment and are consumed by phagocytes to give the
appearance of clear spaces filled with cellular debris.aj
38.38. Microphotograph shows a later stage of apoptosis(A) in
epithelial cells of endometrial glands at the beginning of
menstruation. aj
39.39. Cell turnover in crypts of colonic epithelium occurs by
apoptosis. aj
40.40. Corpus luteum, formed from an ovarian follicle after
discharge of an ovum. Unfertilised ovum, the corpus luteum
will involute, a process that involves progressive death of its
constituent cells, leaving a fibrotic scar known as a corpus
albicans. In this micrograph several apoptotic cells AC can
be identified by their condensed nuclei and eosinophilic
cytoplasmaj

41.41. Apoptosis of two neutrophils in normal anticoagulated

blood during standing at room temperature. Nuclear
condensation and fragmentation are evident. A normal
neutrophil is also present. aj
42.
42. aj
43.43. Apoptosis of an epidermal cell in an immune
reaction(GVHD). The cell is reduced in size and contains
brightly eosinophilic cytoplasm and a condensed nucleusaj
44.
44. Councilman hyaline body, is an eosinophilic
globule often surrounded by normal parenchyma found in
the liver of individuals suffering from viral hepatitis, yellow
fever, or other viral syndrome. It represents a
hepatocyte that is undergoing apoptosis.aj
45.
45. Erythema Multiforme (EM) is a hypersensitivity
reaction usually caused by infections, mostly Herpes simplex
virus. Keratinocytes show apoptosis. aj
46.46. Dysregulated apoptosis -too less -too much aj
47.47. Defective (less than normal)apoptosis in cancer Cancer
cells resist apoptosis by Gaining mutated TP53 Producing
more anti-apoptotic factors TP53 In normal conditions
causes DNA repair or if damage is irrepairable it causes
apoptosis. In most human cancers the causative oncogenic
viruses inactivate TP53 by binding to it. Due to this excess
cell proliferation forms a tumor mass. aj
48.48. Excessive apoptosis Neurodegenerative diseases
Apoptosis of specific set of neurons having mutations or
misfolded proteins. Eg. Alzheimers disease, CJ disease,
cystic fibrosis Death of viral infected cells in many viral
infections. aj
49.
49. Apoptosis vs necrosis aj
50.
50. aj
51.
51. New concepts- aj
52.52. Assays for apoptosis Aim- to find the fragmented DNA
Demonstration of nucleosomal laddering which is
diagnostic of DNA fragmentation This method uses
electrophoresis TUNEL assay This method uses flow

cytometry Gold standard for detecting apoptosis in fixed

tissue is electron microscopy aj
53.
53. S u m m a r y aj