Apoptosis:

Programmed Cell Death

Molecular Biology
Faculty of Pharmacy
Universitas Muhammadiyah Purwokerto
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
INTRODUCTION

Cell death by injury

-Mechanical damage
-Exposure to toxic chemicals
Cell death by suicide
-Internal signals
-External signals
Conted…..

Apoptosis or programmed cell death, is carefully

coordinated collapse of cell, protein degradation,
DNA fragmentation followed by rapid engulfment
of corpses by neighbouring cells. (Tommi, 2002)

Essential part of life for every multicellular

organism from worms to humans. (Faddy et al.,1992)

Apoptosis plays a major role from embryonic

development to senescence.
 Cell death Mechanism
Apoptosis Autophagy Nekrosis Senescence Mitotic
catastrophe
Perubahan
morfologi sel
Membran sel Blebbing, integritas Blebbing Kehilangan Flattening, ukuran sel ?
membran masih integritas membesar
terjaga membran
Nukleus Kondensasi Kondensasi Degradasi DNA Akumulasi Missegregasi
kromatin, DNA kromatin pada acak heterochromatin foci kromosom selama
laddering, sebagian populasi sitokinesis,
fragmentasi mikronukleus
nukleus
Sitoplasma Terbentuk fragmenPeningkatan Pembesaran Granulasi sel ?
sel (apoptotic
jumlah sel yang organela sel
body), mengalami
depolimerisasi autofagi, degradasi
sitoskeleton badan golgi,
poliribosom, dan
reseptor estrogen
Respon Penekanan sistem ? Stimulasi sistem ? ?
imunologi imun, engulfment imun, inisiasi
sel carcass pertumbuhan sel
dan perbaikan
jaringan
 Necrosis Apoptosis
• Accidental death • Programmed death
• Severe & sudden injury
ischaemia, physical or • Process is more subtle, and
chemical trauma more physiologically
• Cellular and organelle determined.
swelling
• Random spillage of • Cell shrinkage
cellular content • Plasma & nuclear membrane
• Inflammatory response blebbing
• Major site of damage • Production of membrane
enclosed “apoptotic
plasma membrane
•Clear by macrophages

•No inflammatory response”

 Necrosis vs. Apoptosis
Necrosis
• Cellular swelling
• Membranes are broken
• ATP is depleted
• Cell lyses, eliciting an
inflammatory reaction
• DNA fragmentation is
random, or smeared
• In vivo, whole areas of the
tissue are affected
Apoptosis
 Cellular condensation
 Membranes remain intact
 Requires ATP
 Cell is phagocytosed, no
tissue reaction
 Ladder-like DNA
fragmentation
 In vivo, individual cells appear
affected
NECROSIS Vs APOPTOSIS

Wilde, 1999
 CellsCells are born,
are born, live live
for afor
given
period aofgiven
timeperiod
and then die
of time and then die Bowen,
1998 Bowen, 1998

APOPTOSIS
--- Physiological cell
deathdeath
--- Cell suicide
--- Cell deletion
--- Programmed cell
Why should a cell commit suicide?
Apoptosis is needed for proper development
Examples:
◦ The resorption of the tadpole tail
◦ The formation of the fingers and toes of the fetus

Apoptosis is needed to destroy cells

Examples:
◦ Cells infected with viruses
◦ Cells of the immune system
◦ Cells with DNA damage
◦ Cancer cells
Apoptosis in Development
 Elongation of long
bone
 Amphibian
metamorphosis
18_27_nerve_target.jpg
What makes a cell decide to commit suicide?

Withdrawal of positive signals

examples :
◦ growth factors for neurons
◦ Interleukin-2 (IL-2)

Receipt of negative signals

examples :
◦ increased levels of oxidants within the cell
◦ damage to DNA by oxidants
◦ death activators :
 Tumor necrosis factor alpha (TNF-)
 Lymphotoxin (TNF-β)
 Fas ligand (FasL)
STAGES OF APOPTOSIS

Induction of apoptosis related genes, signal transduction

Sherman et al., 1997

 Apoptosis: Pathways
“Extrinsic Pathway”

Death Death Initiator

Ligands Receptors Caspase 8

Effector
Caspase 3
“Intrinsic Pathway”

DNA Initiator
Mitochondria/
damage Caspase 9
Cytochrome C
& p53
MAJOR PLAYERS IN APOPTOSIS

• Caspases
• Adaptor proteins
• TNF & TNFR family
• Bcl-2 family
Ligand-induced cell death
Ligand Receptor
FasL Fas (CD95)
TNF TNF-R
TRAIL DR4 (Trail-R)
KELUARGA PROTEIN BCl-2
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.
 APOPTOSIS: Signaling & Control pathways I

Externally driven Externally driven

Apoptotic signals Activators of

p53 initiator enzymes
Internally Cytochrome C
driven
Initiator caspases
mitochondrion
6, 8, 9,12
Execution caspases
2, 3, 7

Apoptosis events

Activation
 APOPTOSIS: Signaling & Control pathways II

Externally driven Externally driven

Inhibitors
Apoptotic signals Activators of
p53 initiator enzymes
Internally Cytochrome C
driven
Bcl2
External Survival
Initiator caspases Internal factors
6, 8, 9,12
Execution caspases
2, 3, 7 Inhibitors of
apoptosis
Apoptosis events

Inhibition
 The mitochondrial pathway

DNA Fas Growth factor

damage receptors
Casp8
PI3K
p53 Bid Akt
casp3
Bid
BAD
Bax
Bid casp9 IAPs
Bcl2 Apaf1
ATP
Bax Cyt.C casp3
Smac/
DIABLO
H2O2
AIF
Pollack etal., 2001
 Importance of Apoptosis

• Important in normal physiology / development

– Development: Immune systems maturation,
Morphogenesis, Neural development
– Adult: Immune privilege, DNA Damage and wound
repair.

• Excess apoptosis
– Neurodegenerative diseases

• Deficient apoptosis
– Cancer
– Autoimmunity
Ways to detect Apoptosis
 Morphological evidence (mentioned
above)
 Caspase activation
 DNA fragmentation
 Phosphatidylserine translocation
 TUNEL
 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
 DNA Fragmentation
 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
 Comet assay
 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.

– +
Comet assay
Possible results of a comet assay

Normal nucleus without

fragments
(DNA is not damaged –
mutagenicity excluded)

Two nuclei with DNA

damage
 Detection of Apoptosis by Flow
Cytometry

 Early stage Annexin V/7-AAD(PI)

 Mid stage TUNEL assay

 Late stage < Go/G1 DNA content

PENGAMATAN APOPTOSIS DENGAN ANNEXIN V
DAN PROPIDIUM IODIDE (PI)
Phosphatidylserine Translocation
(Detected by Flow Cytometry)
PENGAMATAN APOPTOSIS DENGAN
ANNEXIN V DAN PROPIDIUM IODIDE (PI)
Apoptosis

TUNEL ASSAY
TdT-mediated dUTP Nick-End Labeling

DNA degradation

Incorporation of fluorescein-12-dUTP to
3’-OH DNA ends using
enzyme Terminal deoxynucleotidyl Transferase (TdT)

****
dUTP
5’ 3’
OH
TUNEL
 Terminal dUTP nick
end labeling
 Detect DNA nicks by
elongating them with
terminal dNTP
transferase
 dUTP-biotin (or other
conjugates) added as
label
Apoptosis (TUNEL) from Rat Lavage Fluid
Control, 11 months Sterling V, 11 months

0.97 % 20.82%

G1
G1

Sub-G1

S
G2M S
G2M

DNA Content DNA Content

 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