APOPTOSIS

Dr.Dinesh T
Junior resident
Department of Physiology
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Discussion headings
Introduction
Etiopathogenesis
Morphological, Biochemical changes
Mechanism Intrinsic & Extrinsic pathway
Disorders of apoptosis
Conclusion
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Introduction

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Apoptosis - Definition
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.

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"We are most grateful to Professor James Cormack of

the Department of Greek, University of Aberdeen, for
suggesting this term. The word "apoptosis" ()
is used in Greek to describe the "dropping off" or "falling
off" 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 "ptosis" (with the "p" silent),
which comes from the same root "to fall", and is already
used to describe the drooping of the upper eyelid
Kerr Wyllie and Currie paper, British Journal of Cancer, 1972 Aug;26(4):23957
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Historical aspects

German scientist Carl Vogt - Principle of apoptosis

(1842).
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 Walther Flemming Process of programmed

cell death (1845).
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 John Foxton Ross Kerr Distinguish

apoptosis from traumatic cell death
(1962).
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Nobel prize in 2002 Sydney Brenner , Horvitz,

John Buston.
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Cell death
mechanisms
Death by
suicide

Death by injury

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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

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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

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Significance of apoptosis

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]

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)

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)

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Etiopathogenesis

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Why should a cell commit suicide?

1. Programmed cell death is as needed for proper normal development

as mitosis is.
Examples:

o The resorption of the tadpole tail in frog .

o The formation of the fingers and toes of the fetus requires the
removal, by apoptosis.
o The sloughing off of the endometrium at the start of menstruation.
o The formation of the proper connections (synapses) between
neurons in the brain.

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 2. Programmed cell death is needed to destroy cells that

represent a threat to the integrity of the organism.
Examples:
o Cells infected with viruses
o Cells of the immune system
o Cells with DNA damage
o Cancer cells (Uncontrolled proliferated cells)

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Apoptosis in physiologic situations

o
Programmed destruction during embryogenesis
o

Involution of hormone dependent tissues

Cell loss in proliferating cell populations

Elimination of harmful self- reactive lymphocytes

Death of host cells

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Apoptosis in bud
formation during
which many
interdigital cells
die. They are stained
black by a TUNEL
method

Incomplete differentiation
in two toes due to lack of
apoptosis

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Apoptosis: in embryogenesis
Morphogenesis (eliminates excess cells):

Selection (eliminates non-functional cells):

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Apoptosis: in embryogenesis
Immunity (eliminates dangerous cells):
Self antigen
recognizing cell

Organ size (eliminates excess cells):

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Apoptosis: importantance in adults

Tissue remodeling (eliminates cells no longer needed):

Apoptosis
Virgin mammary gland

Late pregnancy, lactation

- Testosterone

Apoptosis
Prostate gland

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Involution
(non-pregnant, non-lactating)

Apoptosis: importantance in adults

Tissue remodeling (eliminates cells no longer needed):

Apoptosis

Resting lymphocytes

+ antigen (e.g. infection)

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- antigen (e.g. recovery)

Apoptosis in pathological conditions

- DNA damage
- Accumulation of misfolded proteins
- Cell death in certain infections
- Pathological atrophy in parenchymal
organs
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Cells of the immune system

CTLs induce apoptosis in each other and
even in themselves.
Defects in the apoptotic machinery is
associated with autoimmune diseases such
as lupus erythematosus and rheumatoid
arthritis.

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Cells infected with viruses

One of the methods by which cytotoxic T
lymphocytes (CTLs) kill virus-infected cells
is by inducing apoptosis

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Cells with DNA damage

Damage to its genome
can cause a cell
to disrupt proper
embryonic
development
leading to birth
defects
to become
cancerous.
Cells respond to DNA
damage by increasing
their production of p53.
p53 is a potent inducer
of apoptosis.
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 Cancer cells
Radiation and chemicals used in cancer therapy
induce apoptosis in some types of cancer cells.

Fig. 1: SC-1 induced apoptosis in stomach carcinoma cells

Left: Before induction
Middle: 24h after induction
Right: 48h after induction sclero dinesh

Morphological & Biochemical changes

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Classic changes
Cell shrinkage
Nuclear fragmentation
Chromatin condensation
Chromosomal DNA fragmentation
Formation of cytoplasmic blebs& apoptotic bodies
Phagocytosis
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Histology
Apoptotic bodies
Round oval mass
of intensely
eosinophillic
cytoplasm
Fragments of
dense nuclear
chromatin
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Bio chemical changes

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Mechanisms of apoptosis

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Caspases

Caspase are Cysteine- Aspartic acid specific proteases that mediates

the events that are associated with programmed cell death.

Their catalytical activity depends on a critical cysteine-residue within a

highly conserved active-site pentapeptide QACRG,

Caspases specifically cleave their substrates after Asp residues.

Caspase- 8, Caspase- 9
acts as an initiator of the caspase activation cascade.

Caspase-3
key effector in the apoptosis pathway, amplifying the signal from
initiator caspases and signifying full commitment to cellular
disassembly.
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Initiation
Absence of stimuli - hormones, growth factors
Activation of receptors TNF family
Heat ,radiation, chemicals
Genetically programmed events

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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
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Receptor pathway (physiological):

FAS ligand

Death receptors:
(FAS, TNF-R, etc)

TNF

Death
domains
Adaptor proteins

Pro-caspase 8 (inactive)

Pro-execution caspase (inactive)

MITOCHONDRIA
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Caspase 8 (active)

Execution caspase (active)

Death

Apoptosis triggered by external signals:

the extrinsic or death receptor pathway

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Regulation of apoptosis
Regulatory proteins BCL -2, equivalent
to CED -9
Apoptosis depends on binding of BCL -2
with pro apoptotic and anti apoptotic
proteins.
Situated in the outer mitochondrial
membrane.
Apaf -1 equivalent to CED -4.
Tp 53, caspases, BAX, viruses such as
adeno, papilloma , hepatitis B.
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Intrinsic pathway (damage):

Mitochondria

BAX
BAK
BOK
BCL-Xs
BAD
BID
B IK
BIM
NIP3
BNIP3

Cytochrome c release
Pro-caspase 9 cleavage

Pro-execution caspase (3) cleavage

BCL-2
BCL-XL
BCL-W
MCL1
BFL1
DIVA
NR-13
Several
viral
proteins

Caspase (3) cleavage of cellular proteins,

nuclease activation,
etc.
Death
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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
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Physiological
receptor pathway

Intrinsic
damage pathway

MITOCHONDRIAL SIGNALS

Caspase cleavage cascade

Orderly cleavage of proteins and DNA
CROSSLINKING OF CELL CORPSES; ENGULFMENT
(no inflammation)
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Steps

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Disorders of apoptosis

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Apoptosis: Role in Disease

TOO MUCH: Tissue atrophy
Neurodegeneration
Thin skin
etc

TOO LITTLE: Hyperplasia

Cancer
Athersclerosis
etc

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Apoptosis: Role in Disease

Neurodegeneration
oNeurons are post-mitotic (cannot replace themselves; neuronal stem cell
replacement is inefficient)
oNeuronal death caused by loss of proper connections, loss of proper growth
factors (e.g. NGF), and/or damage (especially oxidative damage).
oNeuronal dysfunction or damage results in loss of synapses or loss of cell
bodies (synaptosis, can be reversible; apopsosis, irreversible)
oPARKINSON'S DISEASE
oALZHEIMER'S DISEASE
oHUNTINGTON'S DISEASE etc.
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Apoptosis: Role in Disease

Cancer
oApoptosis eliminates damaged cells (damage => mutations =>
cancer
oTumor suppressor p53 controls senescence and apoptosis
responses to damage.
oMost cancer cells are defective in apoptotic response(damaged,
mutant cells survive)
oHigh levels of anti-apoptotic proteins
or
oLow levels of pro-apoptotic proteins
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===> CANCER

Apoptosis: Role in Disease

Cancer
Virus associated cancer
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.
Epstein-Barr Virus (EBV), the cause of mononucleosis
and associated with some lymphomas
produces a protein similar to Bcl-2
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).
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Apoptosis: Role in Disease

Cancer
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.
Melanoma (the most dangerous type of skin cancer)
cells avoid apoptosis by inhibiting the expression of the
gene encoding Apaf-1.

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Apoptosis: Role in Disease

Cancer
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).
Some cancer cells, especially lung and colon cancer
cells, secrete elevated levels of a soluble "decoy"
molecule that binds to FasL, plugging it up so it cannot
bind Fas. Thus, cytotoxic T cells (CTL) cannot kill the
cancer cells

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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)
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Apoptosis: Role in Disease

Apoptosis and AIDS
Hallmark- the decline in the number of the
patient's CD4+ T cells (normally about 1000
per microliter (l) of blood).

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In Immune system
o
o
o

Very rarely humans are encountered with

genetic defects in apoptosis.
The most common one is a mutation in the
gene for Fas
mutations in the gene for FasL or even one of
the caspases are occasionally seen.
Autoimmune lymphoproliferative syndrome
or ALPS.
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Conclusion

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Cells are balanced between life and death

DAMAGE

Physiological death signals

DEATH SIGNAL
ANTIAPOPTOTIC
PROTEINS
(dozens!)

PROAPOPTOTIC
PROTEINS
(dozens!)

DEATH

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Thank u.
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