Robbin's Basis

1. Dr. Al-Farah

2. Learning objectives
 Discuss causes, morphological and
biochemical changes, clinic-pathologic
correlations in Apoptosis.
 Summarize the pathways of apoptosis.

4. APOPTOSIS
Programmed, enzyme-mediated
cell death
Apoptosis is a type of cell death that is induced by a
tightly regulated suicide program in which cells destined
to die activate intrinsic enzymes that degrade the cells’
genomic DNA and nuclear and cytoplasmic proteins.

6. APOPTOSIS
1) Cell is broken into apoptotic bodies
(morphologic appearance of membrane-bound
fragments derived from cells, and named after
the Greek designation for “falling off.”).
2) In which membrane remain intact.
3) It become tasty for phagocytosis.
4) Cell death does not illicit inflammatory reaction.

7. APOPTOSIS: Purpose/Causes
Apoptosis occurs in two broad contexts:
 As a normal physiologic processes, and
 As a pathophysiologic processes of cell
loss in many different diseases.

8. Apoptosis in Physiologic Situations
 Death by apoptosis is a normal phenomenon
that serves to eliminate cells that are no
longer needed, or as a mechanism to
maintain a constant number of various cell
populations in tissues.
 In the average adult between 50 and 70 billion
cells die each day by apoptosis.

9. Apoptosis in Physiologic Situations
1) Removal of supernumerary cells (in excess of the required number) during
development.
EXAMPLE: Apoptosis during embryogenesis i.e., implantation, angiogenesis,
organogenesis, developmental involution and metamorphosis.
2) Involution of hormone dependent tissue upon hormone withdrawal
EXAMPLE: menstruation, regression of lactating breast after weaning, prostatic
atrophy after castration.
3) Cell turnover in proliferating cells
EXAMPLE:
- Immature lymphocytes in bone marrow and thymus that fail to express
useful antigen receptor.
- Epithelial cells in intestinal crypts to maintain constant number.
- B Lymphocytes in germinal center.

10. 4) Elimination of potentially harmful self-reactive lymphocytes
EXAMPLE: Induction of Tolerance in Thymus or periphery
5) Death of host cells that have served their useful purpose
EXAMPLE: Neutrophils in an acute inflammatory response, and
lymphocytes at the end of an immune response.
Apoptosis in Physiologic Situations

11. Apoptosis in pathologic conditions
 Apoptosis eliminates cells that are injured
beyond repair without eliciting a host reaction

12. Apoptosis in pathologic conditions
1) DNA damage: If repair mechanism for damaged
DNA cannot cope with the injury, the cell’s intrinsic
mechanism induces apoptosis.
EXAMPLE: Radiation and cytotoxic anticancer drugs
can damage DNA
2) Accumultaion of misfolded protein: Cell death
triggered by improperly folded intracellular proteins
and the subsequent endoplasmic reticulum (ER)
stress response.

13. Apoptosis in pathologic conditions
3) Cell death in certain infections:
EXAMPLE:
a) In viral infections: loss of infected cells by apoptosis induced
by virus (adenovirus and HIV).
b) By host immune response: In viral hepatitis through cytotoxic
cells.
c) By cytotoxic lymphocytes: In tumor and cellular rejection of
transplant same T- cell mediated (cytotoxic T- cell ).
4) Pathologic atrophy in parenchyma of organ after duct
obstruction
EXAMPLE:
Duct obstruction by stone in pancreas, parotid gland and

14. Apoptosis: Morphologic changes
1) Cell shrinkage:
 Cell size is reduced, the cytoplasm
is dense (organelles tightly packed)
and eosinophilic.
2) Chromatin condensation:
 Most characteristic feature.
 Aggregate peripherally under the
nuclear membrane into dense
masses of various shapes and sizes.
 Nucleus may breakup into two or
more fragments.

16. 3) Formation of cytoplasmic bleb and apoptotic
bodies.
4) Phagocytosis of apoptotic bodies by
macrophages.
 Plasma membrane remain intact during
apoptosis until last stage.
 High speed and efficiency.
 Does not illicit inflammation.
Apoptosis: Morphologic changes

17. APOPTOSIS: Formation of cytoplasmic bleb

18. Summary of Morphological events
 Cells shrink and become detached from adjoining cells
 Cytoskeleton collapses.
 Mitochondria remain intact.
 Plasma membrane develops bubbles (blebs) on surface.
 Nucleus and chromatin condense.
 Chromatin aggregates at periphery of nucleus
 Nuclear envelope disintegrates
 DNA fragmentation
 Budding off and breakage into small membrane wrapped
fragments - apoptotic Bodies

19. The three main players
 Regulating gene - p53 gene
 Protein family - Bcl proteins
 Family of enzymes - Caspases
Common intracellular machinery for
apoptosis

20. Apoptosis: p53 gene and p53 protein
• p53 is tumour suppressor gene
• Active gene product p53 produced in response to
DNA and cell damage
• Prevents cell completing cell cycle
• If damage is minor - allows repair
• If major - induces apoptosis
• Complex mechanisms

21. Apoptosis: BCL gene family
Large family of proteins
There are more than 20 members of the BCL
family, which can be divided into three groups
based on their function and the BCL2 homology
(BH) domains they possess.
1. Anti-apoptotic
2. Pro-apoptotic
3. Sensors (Regulated apoptosis initiators)

23. Apoptosis: BCL family Anti-apoptotic
 Cell survival stimuli induce the expression of anti-apoptotic BCL
proteins.
 BCL2, BCL-XL, and MCL1 are the principal members.
 Possess four BH domains (called BH1-4).
 BCL2 was the first apoptosis-related gene that was recognized to play a role
in tumorigenesis, and indeed, BCL-2 is overexpressed in a variety of
cancers, contributing to cancer cell survival through direct inhibition of
apoptosis.
 Its product is an integral membrane protein (called Bcl-2) located in
the membranes of the endoplasmic reticulum (ER), nuclear envelope,
and in the outer membrane of the mitochondria.
 BCL-2 proteins maintain mitochondrial membrane integrity and
prevent leakage of mitochondrial proteins that can trigger apoptosis
(e.g., cytochrome c).

24.  BAX and BAK – are the principal members.
 Possess the first three BH domains (BH1-3)
 Activated by damage to DNA, misfolded proteins,
viral infections, and other injurious events.
 On activation, BAX and/or BAK oligomerize within the
outer mitochondrial membrane and enhance its
permeability through forming channels in the
mitochondrial membrane that cause leakage of
cytochrome c into the cytosol.
Apoptosis: BCL family Pro-apoptotic

25.  BAD, BIM, BID, Puma, and Noxa – are the principal
members.
 Possess only one BH domain, the third of the four BH
domains, and hence called BH3-only proteins.
 Sensors of cellular stress and damage; when upregulated
and activated, they can initiate apoptosis.
Apoptosis: BCL family Sensor genes

27. Apoptosis: Caspases
 Caspases term is derived from cysteine-dependent aspartate-specific proteases
(enzymes).
 Caspases exist in inactive form , they must undergo cleavage to become active.
10 members – divided into two groups:
1. Inflammatory caspases: 1, 4 and 5
 No role in Apoptosis
2. Initiator caspases : 2, 8, 9 and 10
 Provide a link between cell signalling and apoptotic execution by activating
executioner caspases
3. Executioner caspases: 3, 6, 7
 Activated caspases has proteolytic activity, resulting in morphological and
biochemical markers of apoptosis.
 Targets of Executioner caspases: nuclear lamins, DNA (endonucleases),
cytoskeleton, cell-cell adhesion molecules.

28. Apoptosis: Caspases
 Granzyme B released by cytotoxic T lymphocytes which is
known to activate caspase-3 and -7.
 Death receptors (like FAS, TRAIL receptors and TNF
receptor) which can activate caspase-8 and -10; and
 Apoptosome, regulated by cytochrome c and the Bcl-2
family, which activates caspase-9.

30. Regulation of apoptosis
 Factors controlling apoptosis thus include substances
outside the cell and internal metabolic pathways:
 Inhibitors include growth factors, extracellular cell matrix,
sex steroids, some viral proteins.
 Inducers include growth factor withdrawal, loss of matrix
attachment, glucocorticoids, some viruses, free radicals,
ionising radiation, DNA damage, ligand-binding at ‘death
receptors’.

31. Apoptosis results from the activation of enzymes called
Caspases.
The process of apoptosis may be divided into an
1. Initiation phase
Initiation occur through two distinct pathways (stimuli) which
involve different sets of protein.
Control and integration of proapoptotic and antiapoptotic signals.
Selected caspases become catalytically active and unleash a cascade
of other caspases
2. Execution phase
During which the terminal caspases trigger cellular fragmentation.
3. Removal of Dead Cells (Phagocytosis)
Apoptosis: Mechanism

32. Apoptotic transduction pathways
 There are two distinct pathways that converge on
caspase activation:
 Mitochondrial or intrinsic pathway
 Death activator or extrinsic pathway
 The two pathways of apoptosis differ in their induction
and regulation, and both culminate in the activation of
caspases.

33. Apoptosis: Mechanism

34. Apoptosis: Mechanism
Initiation phase: Mitochondrial pathway
Death Signals
 Growth factor withdrawal
 DNA damage (by radiation, UV lights, X-rays,
Chemotherapeutic drugs, toxins, free radicals)
 Protein misfolding (ER stress)

35. Apoptosis: Mitochondrial or intrinsic
pathway
 Mitochondrial pathway – is the major mechanism of apoptosis
 Resulted from increased mitochondrial permeability and release of pro
apoptotic molecules into the cytoplasm.
 Proteins in mitochondria as Cytochrome C (Essential of life).
 Protein in cytoplasm (released into cytoplasm) – initiate suicide program of
apoptosis.
 Release of mitochondrial protein controlled by – balance between the
proapoptotic and antiapoptic members of BCL2 Family
 In order to understand the cascade of events, an understanding of the BCL
family of genes is important.

36. Apoptosis: Mitochondrial or intrinsic pathway
When a cell is deprived of critical survival signals or their DNA is damaged or
misfolded proteins, sensors of damage or stress are activated.
These sensors proteins are also BCL proteins i.e., include BIM, BID & BAD.
This sensor proteins in-turn activate the pro-apototic factors: BAK & BAX.
Activation of BAX and BAK genes produces protein channels in the mitochondrial
membrane that cause leakage of cytochrome C into the cytosol.
Once Cytochrome C released into the cytoplasm it binds with APAF (apoptotic
protease activating factor) to form an apoptosome.
Apoptosome formed causes the activation of an initiator caspase (Caspase-9), which
in turn activates effector caspases (proteases, endonucleases) that mediate the
execution phase.

37. Viable cell Apoptosis

39. Apoptosis: Mechanism
Death activator or extrinsic pathway
 This pathway is initiated by engagement of plasma membrane death receptors.
 Death receptors are members of the tumor necrosis factor (TNF) receptor
family that contain a cytoplasmic domain (called death domain) involved in
protein protein interactions.
 Best known death receptors are
 Type 1 TNF receptor (TNFR1) activated by Tumour necrosis factor alpha (TNF-α)
 Fas (CD95) activated by Fas ligand (CD95)
 TNF-α is an important cytokine that is involved in systemic inflammation, autoimmune disease, and
wasting (cachexia) in cancer. It is primarily produced by macrophages; however, it can also be produced
by T cells, mast cells, endothelial cells, cardiac cells, and neurons, which explains its multiple disease
associations.
 FasL is expressed on T cells that recognize self antigens and on some Cytotoxic lymphocytes that kill
virus-infected and tumor cells.
o Apoptosis can also be induced by cytotoxic T-lymphocytes using the
enzyme granzyme.

40. Apoptosis: Mechanism
Death activator or extrinsic pathway
 Binding of ligand to its specific death
receptor is a pro-apoptotic signal.
 Signals linked to execution phase through
an integration stage.

41. Apoptosis: Mechanism
Death activator or extrinsic pathway
FAS ligand bind to FAS receptor
Form cross link between three or more FAS
receptor (Death inducing signal complex)
cytoplasmic death domains form a binding
site for an adaptor protein called FADD
(Fas-associated death domain)
FADD binds inactive caspase-8 (or caspase-
10) in turn recruit and activate caspase-8
Active caspase-8 initiates the executioner
caspase sequence which mediate the
execution phase of apoptosis

42. Apoptosis: Mechanisms
Execution Phase
Here the 2 initiating pathways converge to a cascade of caspase
activation which mediates the final phase of apoptosis.
The intrinsic mitochondrial pathway activates the initiator caspase-9,
whereas The extrinsic death receptor pathway activates caspase-8
and caspase-10.
Active forms of these caspases trigger the rapid and sequential
activation of the executioner caspases, such as caspase-3 and
caspase-6, which then act on many cellular components.

43. Apoptosis: Mechanisms of Apoptosis
Execution Phase
Activated effector caspases lead to ...
 Digestion of cytoskeleton proteins
 Nucleus and chromatin degradation
 Plasma membrane changes
 Executioner caspases cause inactivation of DNA repair enzymes,
activation of DNAse that degrade DNA and nucleoproteins.
 Caspases also degrade components of the nuclear matrix and
cytoskeleton, leading to fragmentation of cells.

44. Plasma Membrane alteration and recognition by phagocytes:
 Normally, Enzyme system keeps phospholipids
(phosphotidylserine) on inner surface
During apoptosis
 Phosphotidylserine “flips” out and is expressed on the outer
layer of the membrane.
 Macrophage receptors recognise and bind Phosphotidylserine
followed by engulfment of apoptotic bodies.
 This process of apoptotic cell phagocytosis is called Efferocytosis
 Cells that are dying by apoptosis also secrete soluble factors that
recruit phagocytes.
Apoptosis: Mechanisms of Apoptosis

45. Apoptosis: Mechanisms
CONCEPT MAP

47. Apoptosis: Mechanisms of Apoptosis
 Lets see the video

49. Disorders Associated with Dysregulated
Apoptosis
Dysregulated apoptosis (“too little or too much”) has been postulated to explain
aspects of a wide range of diseases.
1. Disorders associated with defective apoptosis and increased cell
survival.
 Malignancy (mutation of TP53 leading accumulation of mutations)
 Autoimmune disorders (failure to eliminate potentially harmful cells, such as
lymphocytes that can react against self antigens, and failure to eliminate dead
cells, a potential source of self antigen)
 Congenital anomalies Failure of apoptosis in these four sites is a factor in the
development of syndactyly (webbed fingers), cleft palate, spina bifida, and bladder
diverticulum (pouch) or fistula (open connection) from the bladder to the umbilical
skin, respectively.

50. • BAX mutation in tumor cells
• p53 mutation
FLIP resembles initiator caspases, but
lacks proteolytic domain, compete
with caspase 8 and caspase 10 for
binding site in the Death inducing
signal complex
Inhibitor of apoptosis (IAP) proteins block the
inappropriate activation of caspases,
including executioners like caspase-3, and
keep cells alive. Mainly act on the intrinsic
pathway
Defective
apoptosis
and
increased
cell
survival.

51. Disorders Associated with Dysregulated
Apoptosis
2. Disorders associated with increased
apoptosis and excessive cell death.
o Neurodegenerative diseases (over-activation of
some caspases such as caspase-3)
o Ischemic injury and
o Death of virus-infected cells in many viral
infections

52. Review question 1
The apoptosis is classified as
a) Programmed cell death
b) Non-programmed cell death
c) Accidental cell death
d) Mitotic cell death

53. Review question 2
In which of the following situations would cells die by
necrosis, not apoptosis?
a) Removal of virus infected cells.
b) Removal of developing neurons which fail to make
profitable connections with other cells.
c) Removal of heart muscle cells damaged by oxygen
depletion following cardiac infarction.
d) Removal of cells with damaged DNA which cannot
be repaired.

54. Review question 3
Apoptosis can’t kill which of the following?
a) Cell infected with viruses
b) Cell with DNA damage
c) Cancer cells
d) Immune cells

55. Identify?

56. Review question 5
Apoptotic bodies can be recognized with the presence of
these on the surface
(a) phosphatidyl tyrosine
(b) phosphatidylinositol
(c) phosphatidylcholine
(d) phosphatidylserine

57. Review question 6
Which of the following are killed by the extrinsic
apoptosis pathway?
a) Virus infected cells.
b) Cells with damaged DNA.
c) Developing nerve cells which fail to make
profitable connections.
d) Irradiated cells

58. Thank you