MOLECULAR BIOLOGY (FAF 2771)

WEEK 13
NOVEMBER 2022

Apoptosis and Cell Death

Prepared by Prof. Edy Meiyanto
Modified by Dr. Muthi Ikawati
 Molecular Biology 3

Map of the topic

Use it as guideline to understand the topic :)

I. Cell death II. Cell death III.Molecular

intro CharacteristicApoptosis mechanism
What Necrosis Autophagy Apoptosis How
Why Major players
Where Injury Suicide Programmed
When
Who

IV. Assays to detect Apoptosis

1. Cytomorphological alterations – microscopy
2. DNA fragmentation
3. Detection of caspases, cleaved substrates, regulators and inhibitors – detected by
Western blot
4. Membrane alteration - phosphatidylserine translocation – detected by flow cytometry)
 I. Cell death: Introduction

What
Why
Where
When
Who
 Molecular Biology 5

I. Introduction – Cell death

• The number of cells in a body of organism is tightly regulated. Its
not only the control over cell division but also on rate of cell death.
• Controlled cell death (apoptosis) is needed for normal
development and good health throughout life along with normal
development and maturation of cells.

INTERESTING FACT!!!

Without apoptosis, 2 tons of bone marrow and lymph nodes and a 16-km intestine
would probably accumulate in a human by the age of 80.
 Molecular Biology 6

The Nomenclature Committee on Cell Death

(NCCD) has formulated guidelines for the
definition and interpretation of cell death.

Different forms of CELL DEATH:

1. Type I cell death or apoptosis, culminating
with the formation of apparently intact small vesicles
(commonly known as apoptotic bodies) that are
efficiently taken up by neighboring cells with
phagocytic activity and degraded within lysosomes.
2. Type II cell death or autophagy,
manifesting with phagocytic uptake and consequent
lysosomal degradation.

RCD : Regulated Cell Death

3. Type III cell death or necrosis, displaying
MPT : Mitochondrial Permeability Transition no distinctive features of type I or II cell death and
LDCD : Lysosome-Dependent Cell Death absence of obvious phagocytic and lysosomal
ADCD : Autophagy-Dependent Cell Death involvement.
ICD : Immunogenic Cell Death

Non-lethal processes which involved in cell death
Cellular senescence à the
cells permanently lose their
proliferative capacity while
remaining viable and
metabolically active.
Mitotic catastrophe à
morphologically defined by
unique nuclear changes,
including multinucleation and
micronucleation of cells that
are unable to complete
mitosis owing to extensive
DNA damage.
Molecular Biology 7
Multiple cases of terminal
differentiation à multiple
components of the signal
transduction cascades that
regulate cell death.
 Molecular Biology 8
What?
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
Necrosis
Cell death by injury à Necrosis
-Mechanical damage
-Exposure to toxic chemicals
Cell death by suicide à Apoptosis
-Internal signals
-External signals
 Molecular Biology 9

Cells are born,

Cells livelive
are born, forfor
a given
period ofa time and then die
given period
of time and then die Bowen,
[Bowen, 1998]
1998

--- Physiological
APOPTOSIS cell
deathdeath
--- Cell suicide
--- Cell deletion
--- Programmed cell
 Molecular Biology 10

Introduction of cancer biology: loss of apoptosis

https://youtu.be/8VSgOeJy4dQ
 Molecular Biology 12
When? where?

What do falling leaves, the development of a mouse’s paw and

a tadpole losing its tail all have in common?
 Molecular Biology 13

Apoptosis definition
• Apoptosis is a process that occurs in multicellular when a cell intentionally
“decides” to die.

• Apoptosis is referred to as “programmed” cell death because it happens

due to biochemical instructions in the cell’s DNA; this is opposed to the
process of “necrosis,” when a cell dies due to outside trauma or
deprivation.

• Like many other complex cellular processes, apoptosis is triggered by

signal molecules that tell the cell it’s time to commit cellular “suicide.”
 Molecular Biology 14
Who?
History of apoptosis
Molecular Biology 15
Molecular Biology 16
 Molecular Biology 17
Why?
Need of apoptosis
Apoptosis is needed for proper development
Examples:
• The formation of the fingers and toes of the
fetus
• The formation of the proper connections
between neurons in the brain

Apoptosis is needed to destroy cells

Examples:
• Cells infected with viruses
• Cells with DNA damage
• Cancer cells
II. Cell death characteristic

Necrosis Autophagy Apoptosis

Injury Suicide Programmed

Molecular Biology 19
 Molecular Biology 20

Apoptosis

https://youtu.be/DR80Huxp4y8
 Characteristics of Apoptosis
Characteristic biochemical changes in cells undergoing apoptosis:
• Chromosomal DNA cleaved into fragments
• Change in the plasma membrane – phosphatidylserine in the outer leaflet
• Loss of electrical potential across the inner membrane of the
mitochondria
• Relocation of cytochrome c from the intermembrane space of the
mitochondria to the cytosol
 Molecular Biology 23

VS

Necrosis Apoptosis
• Accidental death • Programmed death
• Severe & sudden injury ischemia, • Process is more subtle, and more
physical or chemical trauma physiologically determined.
• Cellular and organelle swelling • Cell shrinkage
• Random spillage of cellular content • Plasma & nuclear membrane
• Inflammatory response blebbing
• Major site of damage plasma • Production of membrane enclosed
membrane “apoptotic”
• Clear by macrophage
• No inflammatory response
WATCH ! https://www.youtube.com/watch?v=1vaEVcMfa1E
 Molecular Biology 24

Necrosis vs apoptosis

https://youtu.be/1vaEVcMfa1E
 Molecular Biology 25

Apoptosis and cancer

One primary function of apoptosis is to destroy cells that are
dangerous to the rest of the organism.

A common reason for apoptosis is when a cell recognizes that

its DNA has been badly damaged.

In these cases, the DNA damage triggers apoptosis

pathways, ensuring that the cell cannot become a
malignant cancer.
 Molecular Biology 26
Intrinsic

Extrinsic
 Molecular Biology 27

Internal inducers of
Apoptosis:
• Oncogene Mitochondria

APOPTOSIS PATHWAY
Regulated by Intrinsic Cytochrome c
mitochondria

External inducers of Extrinsic Death receptor

Apoptosis: Fas, TNF alpha
• TNF family
• Growth factor
withdrawn
• Calcium
• Nutrient deprivation
• Toxins
• UV radiation
• Gamma radiation
 Molecular Biology 28

Caspases take major and a central role in apoptotic mechanism. The term caspases is derived from
cysteine-dependent aspartate-specific proteases. Caspases are central to the mechanism of
apoptosis as they are both the initiators and executioners.
 III. Molecular mechanism

How
Major players
How? 30

Molecular mechanism of
apoptosis
There are 2 different mechanisms by which a
cell commits suicide by apoptosis.
- one generated by signals arising within
the cell
- the other triggered by death activators
binding to receptors at the cell surface.
* TNF-a
* Lymphotoxin
* Fas ligand (FasL)
Major players? Molecular Biology 31

Major players in apoptosis

• Caspase
• Adaptor proteins
• TNF & TNFR family
• Bcl-2 Family
 Molecular Biology 32

1. Caspases
• Caspases= Cysteinyl aspartate specific proteases
• A family of intracellular cysteine proteases that play a pivotal role in the
initiation and execution of apoptosis.
• Single chain of inactive proenzymes (zymogen) with 32-56 kDa
• Contains an N-terminal domain, a small subunit and a large subunit
(similar to a ribosome)
• Apoptotic stimulus à activation à substrate cleavage à enzyme
 Molecular Biology 33

Caspase structure

To date, ten major caspases have been identified and broadly categorized into:
a. Signaling or initiator caspases (2, 8, 9, 10)
b. Effector or Executioner caspases (3, 6, 7)
c. Inflammatory caspases (1, 4, 5)
d. The other caspases that have been identified include: Caspases 11, 12, 13, 14
e. Central role in cascade of apoptotic events is played by caspase 3 (CPP32)
 Molecular Biology 34

Two caspase pathways

Extrinsic Pathway Intrinsic Pathway
ê ê
Death Ligand Mitochondria
ê ê
Death Receptors Cytochrome C
ê ê
Initiator Caspase 8 Apoptosome Complex
ê ê
Effector caspase 3 Initiator Caspase 9
ê ê
Cell Death Effector caspase 3
ê
Cell Death
 Molecular Biology 35

2. Adaptor proteins

Apoptotic adaptor proteins play a critical role in regulating pro- and anti-
apoptotic signalling pathways.

Adaptor proteins:
a. FADD (Fas-associated death domain)
b. TRADD (TNF receptor-associated death domain), are recruited to
ligand-activated, oligomerized death receptors to mediate apoptotic
signalling pathways.
 Molecular Biology 36

3. TNF & TNFR family

(ligand-induced cell death)

• “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.
 Molecular Biology 37

4. Bcl-2 family
The control & regulation of apoptotic mitochondrial events occurs through
members of the Bcl-2 family of proteins

1. Anti-apoptotic proteins include Bcl-2,

Bcl-x, Bcl-XL, Bcl-w
2. Pro-apoptotic proteins include Bax,
Bak, Bid, Bad, Bim, Bik
3. 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 à INSTRINSIC
PATHWAY
 38

BCL-2 homology domain

 Molecular Biology 39

Cytochrome C & apoptosome

Cytochrome c is an abundant protein of the mitochondrial inner

membrane, and acts as an electron transport intermediate.

During apoptotic activation lead to alterations in permeability of the

mitochondrial membrane pore proteins and release of cytochrome c.

Cytochrome c binds and activates Apaf-1 as well as procaspase-9,

forming an “apoptosome”.
 Molecular Biology 40

Extrinsic pathway
 Molecular Biology 41

Intrinsic pathway
 Molecular Biology 42

Intrinsic pathway

a. The stimuli that initiate the intrinsic pathway produce intracellular signals
such as radiation (DNA damage), absence of certain growth factors,
hormones and cytokines.
b. All of these stimuli cause changes in the mitochondrial outer membrane
permeabilization (MOMP)
c. Release of pro-apoptotic proteins such as cytochrome c, Smac/DIABLO,
AIF, endonuclease G and CAD from the intermembrane space into the
cytosol.
d. Cytochrome c binds and activates Apaf-1 as well as procaspase-9,
forming an “apoptosome”.
e. Caspase-9 activation, subsequent caspase-3 activation and cell death.
 Molecular Biology 43
APOPTOSIS TARGET DRUGS
 IV. Assays to detect apoptosis

1. Cytomorphological alterations à
microscopy
2. DNA Fragmentation -- electrophoresis
3. Detection of caspases, cleaved substrates,
regulators and inhibitors – western blot
4. Membrane alteration - Phosphatidylserine
Translocation (Detected by Flow Cytometry)
 Molecular Biology 45

Assays to detect apoptosis

Apoptosis assays, based on methodology, can be classified into six major
groups:
1. Cytomorphological alterations (morphological evidence)
2. DNA fragmentation (TUNEL, Flowcytometry, microscopy)
3. Detection of caspases, cleaved substrates, regulators and inhibitors
4. Membrane alterations (phosphatidylserine traslocation)
5. Detection of apoptosis in whole mounts
6. Mitochondrial assays
 Molecular Biology 46

1. Cytomorphological alterations à microscopy

The evaluation of hematoxylin and eosin-stained tissue
sections with light microscopy does allow the visualization
of apoptotic cells.

This method detects the later events of apoptosis.

TEM is considered the gold standard to confirm apoptosis:

1. Electron-dense nucleus
2. Nuclear fragmentation
3. Disorganized cytoplasmic organelles
4. large clear Vacuoles
5. Intact cell membrane
6. Blebs at the cell surface

Other staining methods also categorized in morphological

assay, including acridine orange.
 Molecular Biology 47

2. DNA fragmentation

• In normal cells, DNA are wired around

protein spindle called histones
• DNA and histones form units called
nucleosomes
• In apoptotic cells, cleaved by DNase,
nucleosomes are cut loose, like
beads come off a string
 Molecular Biology 48

DNA fragmentation - electrophoresis

• DNA of apoptotic cells subject to

electrophoresis
• The DNA in nucleosomes cut loose
have lower MW than intact DNA, thus
move faster in electrophoresis
• Result in a “ladder” in the gel
 49

DNA fragmentation - electrophoresis Normal nucleus without

fragments
(Comet assay) (DNA is not damaged –
mutagenicity excluded)
• DNA fragments are released from nuclei using
electrophoresis
• Isolated nuclei are mounted into electrophoretic gel – after
electrophoresis are stained with fluorescent dye.
• If DNA fragments are present a „comet tail“ is present
observed in the vicinity of the nuclei.

– +
Two nuclei with DNA
damage
 Molecular Biology 50

DNA fragmentation – TUNEL ASSAY

TdT-mediated dUTP Nick-End Labeling
• Terminal dUTP nick end labeling
• Detect DNA nicks by elongating
them with terminal dNTP
transferase
• dUTP-biotin (or other conjugates)
added as label
 Molecular Biology 51
Apoptosis (TUNEL) from Rat Lavage Fluid
Control, 11 months Sterling V, 11 months

Anti-BUDR Antibody

Anti-BUDR Antibody
0.97 % 20.82%

(TUNEL)

(TUNEL)
G1
G1
Cell number

Cell number
Sub-G1

S
G2M S
G2M

DNA Content DNA Content

 Molecular Biology 52

3. Detection of caspases, cleaved substrates,

regulators and inhibitors – Western blot

• Caspase-3 cleaves many cellular proteins

including PARP (Poly (ADPRibose) Polymerase).
PARP is a 116 kDa nuclear protein which is
strongly activated by DNA strand breaks .

• PARP plays a role in DNA repair as well as in

other cellular processes, including DNA
replication, cell proliferation and differentiation.

• During apoptosis,caspase-3 and -7, cleave PARP

to yield an 85 kDa and a 25 kDa fragment. PARP
cleavage is considered to be one of the classical
characteristics of apoptosis.
 Molecular Biology 53
SEMA3B induces caspase-3-dependent apoptosis and VEGF165 antagonizes
this effect

Castro-Rivera E et al. PNAS 2004;101:11432-11437

©2004 by National Academy of Sciences

 Molecular Biology 54

4. Membrane alteration - phosphatidylserine translocation

(detected by flow cytometry)
• In healthy cells, phosphatidylserine (PS), an anionic
phospholipid, is actively translocated to the inner leaflet of
the cell membrane.

• During apoptosis, this distribution is randomized, resulting

in the appearance of PS on the outer leaflet.

• Exposed PS can be detected by the calcium dependent

binding of Annexin V.
 Molecular Biology 55

Annexin V/ PI staining
1. During the process of apoptosis, one of the earliest events is Externalization
of Phosphatidylserine (PS) from the inner to the outer plasma membrane of
apoptotic cells.
2. These cells can be demonstrated by bound with Fluorescein isothyocyanate
(FITC)-labeled Annexin V and detected with fluorescent microscopy.
3. The vital dye propidium iodide (PI) should be used in combination of annexin
V that help in distinguish viable , apoptotic & necrotic cell populations at the
same time.

4 3

1 2
 Molecular Biology 56

Summary
Let's recall your understanding. Can you briefly tell each keyword?

I. Cell death II. Cell death III.Molecular

intro CharacteristicApoptosis mechanism
What Necrosis Autophagy Apoptosis How: intrinsic/ extrinsic pathway
Why Injury Suicide Programmed Major players:
Where -Caspase
When
Who -Adaptor proteins
-TNF & TNFR family
-Bcl-2 Family
IV. Assays to detect Apoptosis -Cytochrome C & apoptosome
1. Cytomorphological alterations – microscopy
2. DNA fragmentation
3. Detection of caspases, cleaved substrates, regulators and inhibitors – detected by
Western blot
4. Membrane alteration - phosphatidylserine translocation – detected by flow cytometry)
 Molecular Biology 57

QUICK QUIZ
 Molecular Biology 58

1. Which of the following would you NOT expect to

trigger apoptosis?

A. Damage to a cell’s DNA

B. Long-term oxygen deprivation
C. An organism moving to a new stage of its life cycle,
rendering some cells obsolete
D. None of the above
 Molecular Biology 59

2. Which of the following might occur if a mutation

made apoptosis impossible?

A. The nervous system might not develop properly

B. Cancer might become much more likely
C. An insect might not be able to undergo
metamorphoses
D. All of the above
 Molecular Biology 60

3. What is the difference between the extrinsic and

intrinsic pathways of apoptosis?

A. The extrinsic pathway is triggered by a signal from

outside the cell, while the intrinsic pathway is triggered by
events inside the cell.
B. The extrinsic pathway has more steps because the
signal must be relayed from the cell membrane.
C. The extrinsic pathway activates BAK and BAX, while
the intrinsic pathway does not.
D. A and B
 Molecular Biology 61

References

1. Molecular Biology of The Cell. New York: Garland Science.

2. Galluzzi L, Vitale I, Aaronson SA, Abrams JM, Adam D, Agostinis P, et al.
Molecular mechanisms of cell death: recommendations of the Nomenclature
Committee on Cell Death 2018. Cell Death Differ. 2018 Mar;25(3):486–541.
3. Cavalcante GC, Schaan AP, Cabral GF, Santana-da-Silva MN, Pinto P, Vidal
AF, et al. A Cell’s Fate: An Overview of the Molecular Biology and Genetics of
Apoptosis. IJMS. 2019 Aug 24;20(17):4133.
edy_meiyanto@ugm.ac.id
muthi_ikawati@ugm.ac.id