Apoptosis

Dr. K.L.Krishna

07/30/21 JSS College of Pharmacy, Mysuru

 Apoptosis – Programmed Cell Death
• (True/False) In adult tissues cell death exactly balances cell division
• In apoptosis the cell destroys itself from within and avoids leakage of the cell contents into
the extracellular space. Why do you think that this occurs via a different mechanism than in
necrosis?
• What are some signals that indicate to a cell that apoptosis needs to occur? Where do these
signals come from?
• What are some cellular components involved in the apoptotic pathway?
• What is the difference between a mitogen, a growth factor, and a survival factor?
• In what phase of the cell cycle do cells exit to undergo apoptosis?
• What effects do telomeres and telomerase have on cell aging and death? If you could turn
on telomerase activity in all of our cells, would it prevent aging?
• Do the following types of cells exist in humans?
– Cells that do not grow and do not divide
– Cells that grow, but do not divide
– Cells that divide, but do not grow
– Cells that grow and divide

07/30/21 JSS College of Pharmacy, Mysuru

• In humans, the rate of cell growth and cell death is balanced
to maintain the weight of the body.

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 Cell Death
• The body is very good at maintaining a constant
number of cells. So there has to exist
mechanisms for ensuring other cells in the body
are removed, when appropriate.
• Two forms
– Apoptosis - suicide - programmed cell death
– Necrosis - killing - decay and destruction

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Death by Injury vs. Death by Suicide
(Necrosis vs. Apoptosis)

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Necrosis
Trauma (toxic chemicals, mechanical injury, heat, hypoxia)
Loss of ability to regulate internal environment
Ca2+ influx accompanied by swelling
Alteration of protein activity
calpain
cathepsin
caspase
Production of toxic compounds
(activation of cyclooxygenases)
arachadonic acid
prostaglandins
eicosanoids

Inflammation
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 INTRODUCTION
Cell death by injury
-Mechanical damage
-Exposure to toxic chemicals
Cell death by suicide
-Internal signals
-External signals

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Apoptosis or programmed cell death, is carefully
coordinated collapse of cell, protein degradation ,
DNA fragmentation followed by rapid engulfment of
corpses by neigh-bouring cells.

Essential part of life for every multicellular organism

from worms to humans.

Apoptosis plays a major role from embryonic

development to senescence.

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• Apoptosis = “normal” or “programmed” cell death

• Necrosis = “accidental” or “ordinary” cell death

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• Apoptosis or programmed cell death (PCD) is a mode of cell
death that occurs under normal physiological conditions and
the cell is an active participant in its own demise (“cellular
suicide”).

• It is important for the development of multicellular organism

(embryonic development) and homeostasis of their tissues
(adult).

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Apoptosis results in a quick and clean cell death, without damaging its
neighbours, or eliciting an immune response. Every cell is equipped with
the ‘cell death pathway’. Apoptosis is an intracellular proteolytic
pathway. The DNA is broken into small 200 bp units.

18_20_Apoptosis_.jpg

The cytoplasm shrinks. The mitochondria release cytochrome c. The

outer surface of the plasma membrane gets coated with a different
sugar - one that macrophages can sense and phagocytose.

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Apoptosis is a beneficial and important phenomenon:
 In embryo
1. During embryonic development, help to digit formation.

•Lack of apoptosis in humans

can lead to webbed fingers
called “ syndactyly ”.
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2. Normal event in development of the nervous system

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Apoptosis is a beneficial and important phenomenon:
 In adult
•Normal cell turn over
•Tissue homeostasis
•Induction and maintenance of immune tolerance
•Development of the nervous system
•Endocrine-dependent tissue atrophy
•Elimination of activated, damaged and abnormal cells

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Apoptosis is a beneficial and important phenomenon:
 In animals

Embryonic Mouse Paw

Embryonic Chicken Foot

Tail absorption of the tadpole

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• Apoptosis is the physiological cell death which unwanted or
useless cells are eliminated during development and other
normal biological processes.

• Necrosis is the pathological cell death which occurs when

cells are exposed to a serious physical or chemical insult
(hypoxia, hyperthermia, ischemia).

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• There are many observable morphological and biochemical
differences between necrosis and apoptosis:
 Morphological features
Necrotic cells Apoptotic cells
 Volume enlargement  Volume reduction 
Swelling of cytoplasm  Shrinking of cytoplasm
& mitochondria  No loss of membrane integrity
 Loss of membrane integrity
 No vesicle formation  Formation of apoptotic bodies
 Condensation of chromatin &
DNA fragmentation
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 Oncosis and Necrosis:
Unregulated Cell Death Due to Injury
Cell Swells (Oncosis)
Nucleus Swells
Disruption of Organelles and
Rupture/Release of Contents
Contents Released into
Extracellular Space
07/30/21
Apoptosis:
An Active Regulated Process
DNA Fragmentation
Chromatin Condensation
Fragmentation of Nucleus
Cell Shrinks
Formation of Membrane Enclosed
Fragments called Apoptotic Bodies
Recognition and Engulfment
by Phagocytic Cells
or Neighboring Cells
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JSS College of Pharmacy, Mysuru
 Biochemical features
Necrotic cells
 Loss of regulation of ion homeostasis
 No energy requirement (passive process, also occurs at 4 °C)

Apoptotic cells
 Tightly regulated process
 Energy(ATP)- dependent (active process, doesn’t occur at 4 °C)
 Release of various factors into cytosol by mitochondria
 Activation of caspase cascade

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 Physiological significance
Necrosis
 Affects groups of contiguous cells
 Evoked by non-physiological disturbances (lytic viruses, hypoxia)
 Phagocytosis by macrophages
 Significant inflammatory response
Apoptosis
 Affects single cells or small clusters of cells
 Induced by physiological stimuli(lack of growth factors, DNA damage)
 Rapidly phagocytized by adjacent epithelial cells or macrophages
 No inflammatory response
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JSS College of Pharmacy, Mysuru
 Main Pathways Regulating Caspase
Activation During Apoptosis

Intrinsic Pathway- Mitochondrial Mediated

Major Pathway in Mammalian Cells
– Outer Mitochondrial Membrane Permeabilization (MOMP)
– Release of Cytochrome C from Mitochondrial Intermembrane
Space into Cytosol
– Apoptosome Formation- Activation of Initiator Caspase
– Effector Caspases Activated

Extrinsic Pathway- Signaling through Death Receptors

– Ligand Bound Death Receptors
– Adaptor Protein Association
– Initiator Caspase Recruitment and Activation
– Effector Caspases Activated

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JSS College of Pharmacy, Mysuru
JSS College of Pharmacy, Mysuru
Caspases= Cysteinyl aspartate specific proteases

• A family of intracellular cysteine proteases that play a pivotal

role in the initiation and execution of apoptosis.

• At least 14 different members of caspases in mammalian cells

have been identified

• All are synthesized as inactive proenzymes (zymogen) with

32-56 kDa
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JSS College of Pharmacy, Mysuru
JSS College of Pharmacy, Mysuru
 Caspases
• Proteins which degrade other proteins are
employed by apoptosis - caspases
• Made as inactive precursors - procaspases
• These are activated by other proteins when
the right signal is received
• One caspase cleaves the lamin proteins
resulting in the irreversible breakdown of
the nuclear membrane.

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18_21_proteolytic_cas.jpg

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18_22_Bcl_2_family.jpg

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 The Morphological Changes of Apoptosis Are
Orchestrated by Caspases
Cysteine Proteases that cleave at Aspartic Acid Residues

Activate Apoptosis by Cleaving Specific Substrates

Present but inactive in cells

Two Main Types of Caspases:

1) Initiators- Need to dimerize to become active “induced proximity”

2) Executioner (Effector) - Need to be proteolytically cleaved to become active

- Cleavage is usually Mediated by Initiator Caspases

Zymogens

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Caspase Activation Amplification Cascade
Once Executioners are Activated their
Key Targets of Proteolysis Include:

1)An Inhibitor of a DNAse-

Leads to Fragmentation of DNA

2)Nuclear Lamins-
Leads to Fragmentation of Nucleus

3)Other Cytoskeletal Associated Proteins-

Leads to Disruption of Cytoskeleton and
Cell Fragmentation

4)Additional Caspases

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• “Death receptors” that are members of the tumor necrosis
factor (TNF) receptor superfamily.

• Death receptors have a cytoplasmic domain of

about 80 amino acids called the “death domain”.

• This death domain plays a critical role in transmitting the

death signal from the cell surface to the intracellular signaling
pathways.

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 The best characterized receptors & ligands corresponding
death include:
Receptors Ligands
FasR (CD95/APO1) FasL
DR3 Apo3L
DR4 (TRAIL-R1) Apo2L
DR5 (TRAIL-R2) Apo2L
TNFR1 TNF-α
TNFR2 TNF-ß

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JSS College of Pharmacy, Mysuru
 Binding of trimeric FasL to Fas
 Trimerization and clustering of Fas
 Recruitment of Fas-associated death
domain (FADD) to Fas
 Recruitment of caspase-8 to FADD
 Formation of Death-Inducing Signaling
Complex (DISC )
 Activation of caspase-8 (autoactivation)
 Activation of effector caspases
 Apoptosis

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 Extrinsic Pathway of Apoptosis Activation:
Signaling through the Death Receptors

Ligand Bound Death Receptors Target cells :

Adaptor Protein and Viral Infected Cells or

Procaspase Recruitment Cancer Cells

Initiator Caspase Activation
Effector Caspases Activated
Removal of Excess
Lymphocytes after
Infection
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 Cancer

Cancer is a Disease of Cells that Proliferate at Inappropriate Times and Locations in

the Body.

Tumors (Neoplasms) - Masses of cells derived from a single abnormally proliferating

cell. Tumors are Clonal

1. Benign- Noninvasive, Do not affect other tissues

2. Malignant- Cancerous, Locally Invasive and May Spread

Tumors are classified by cell type from which they arise.

1. Carcinoma- 90% of human cancers- Malignacy of Epithelial Cells
2. Sarcomas – Rare, Solid tumors of connective tissue, such as bone, muscle,
cartilage, and fibrous tissue.
3. Leukemias and Lymphomas- 7% of cancers, Blood forming cells and cells of immune
system
4. Neuroectodermal- Cells of central or peripheral nervous system

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JSS College of Pharmacy, Mysuru
• The stimuli that initiate the intrinsic pathway produce
intracellular signals such as radiation (DNA damage), and
cytokines absence of certain growth factors, hormones.
• All of these stimuli cause changes in the mitochondrial outer
membrane permeabilization (MOMP)
• Release of pro-apoptotic proteins such as cytochrome c,
Smac/DIABLO, AIF, endonuclease G and CAD from the inter-
membrane space into the cytosol.
• Cytochrome c binds and activates Apaf-1 as well as
procaspase-9, forming an “apoptosome”.
• Caspase-9 activation, subsequent caspase-3 activation and
cell death.

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JSS College of Pharmacy, Mysuru
• The control & regulation of apoptotic mitochondrial events
occurs through members of the Bcl-2 family of proteins
 Anti-apoptotic proteins include Bcl-2, Bcl-x, Bcl-XL, Bcl-w
 Pro-apoptotic proteins include Bax, Bak, Bid, Bad, Bim, Bik

 The main mechanism of action of the Bcl-2 family of proteins

is the regulation of cytochrome c release from the
mitochondria via alteration of mitochondrial membrane
permeability.

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Intrinsic Pathway of Apoptosis Activation

MOMPs

cytochrome c Release

Apoptosome Formation:
Adaptor (Apaf1), dATP
cytochrome c and
procaspase complex

Association of Adaptor
with Procaspase allows
Procaspase self cleavage

Active Initiator Caspase

Cleaves Effector
Caspases
Which now Cleave
Targets
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 Critical Regulators of Cell Death

Bcl-2 Family – Regulate whether MOMPs Occurs

Anti-Apoptotic Factors - Death Inhibitors
A) Function to Inhibit MOMPs by Pro Apoptotic Factors

Pro-Apoptotic Factors- Death Activators

A) Bind and inhibit Death Inhibitors
B) Directly cause Permeabilization of MOM to
Stimulate Release of Cytochrome C ( BAX AND BAK)

IAP Family (Inhibitor of Apoptosis)

Bind Procaspases prevent activation
Bind Caspases and inhibit Activity

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 Survival Factor Signaling is
Required to Prevent Apoptosis

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 Programmed Cell Death
in Neuronal Development

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 Survival Factors Signaling Can Function to
Keep Anti-Apoptotic Factor Bcl-2 Active

No Survival Signal
Bcl-2 Complexes with
Bad
Can’t prevent
BAK and BAX
Mediated
MOMPs JSS College of Pharmacy, Mysuru
Aberrant cell death can lead to many human diseases:

 Decreased apoptosis Cancer, Autoimmune disorders

 Excessive apoptosis Neurodegenerative and

immunodeficiency (AIDS) disorders , Ischemia

NOTE: Properties of carcinogenic agents (chemical agents as well as

radiations) are the growth-inhibition power and the ability to induce
cell death. These properties are widely used in anticancer chemo- and
radiotherapies

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(Hyperplasia)

(Tissue atrophy)

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