Apoptosis

Apoptosis

SUBMITED BY

AMIT KUMAR

MPHARM (I Year)

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In humans, the rate of cell growth and cell death is balanced to

maintain the weight of the body.

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Life cannot exist without cellular death

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Cell death can occur via several processes (about 11 type):

1. Apoptosis

2. Necrosis

3. Autophagy

4. Entosis

5. Oncosis

6. Pyroptosis

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Apoptosis = “normal” or “programmed” cell death

Apoptosis is the physiological cell death which

unwanted or useless cells are eliminated during

development and other normal biological processes.

Necrosis = “accidental” or “ordinary” cell death

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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1) Cellular condensation

2) Membranes remain intact

3) Requires ATP

4) Cell is phagocytosed, no tissue

reaction

5) Ladder-like DNA

fragmentation

6) In vivo, individual cells appear

affected

1) Cellular swelling

2) Membranes are broken

3) ATP is depleted

4) Cell lyses, eliciting an

inflammatory reaction

5) DNA fragmentation is

random, or smeared

6) In vivo, whole areas of the

tissue are affected

Necrosis Apoptosis

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The word ‘‘apoptosis’’ comes from the

ancient Greek, meaning the:

‘‘falling of petals from a flower’’ or

‘‘of leaves from a tree in autumn’’

In 1964 Lockshin, study on programmed cell death.

The term apoptosis (a-po-toe-sis) was first used in a

now-classic paper by Kerr et al 1972 to describe a

morphologically distinct form of 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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1) Cell shrinkage

2) Organelle reduction

3) Mitochondrial leakage

4) Chromatin condensation

5) Nuclear fragmentation

6) Membrane blebbing & changes

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Mitochondrial leakage

Organelle

reduction

Cell

shrinkage

Nuclear

fragmentation

Chromatin condensation

Membrane blebbing & changes

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Characteristic biochemical changes in cells undergoing

apoptosis

1)Chromosomal DNA cleaved into fragments

2)Change in the plasma membrane – phosphatidylserine

in the outer leaflet

3)Loss of electrical potential across the inner membrane of the

mitochondria

4)Relocation of cytochrome c from the intermembrane space of

the mitochondria to the cytosol

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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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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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In animals

Embryonic Chicken Foot

Embryonic Mouse Paw

Tail absorption of the tadpole

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APOPTOSIS

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Extrinsic pathway (death receptor- mediated events)

Intrinsic pathway (mitochondria- mediated events)

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Death Receptors

Death Ligands

Adaptor Proteins

Caspaseses

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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 receptors include:

FasR (CD95/APO1) FasL

DR3 Apo3L

DR4 (TRAIL-R1) Apo2L

DR5 (TRAIL-R2) Apo2L

TNFR1 TNF-α

TNFR2 TNF-ß

LigandsReceptors

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Apoptotic adaptor proteins play a critical role in

regulating pro- and anti-apoptotic signalling

pathways

Adaptor proteins;

1)FADD (Fas-associated death domain)

2) TRADD (TNF receptor-associated death domain), are

recruited to ligand-activated, oligomerized death receptors

to mediate apoptotic signalling pathways.

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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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To date, ten major caspases have been identified and broadly categorized into:

Signaling or Initiator caspases

Effector or Executioner caspases

Inflammatory caspases

The other caspases that have been identified include:

Caspases

Central role in cascade of apoptotic events is played by

caspase 3 (CPP32)

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Bcl-2 family proteins

Cytochrome c

Apoptosome

Caspaseses

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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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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”.

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The stimuli that initiate the intrinsic pathway produce intracellular

signals such as radiation (DNA damage), absence of certain growth

factors, hormones and cytokines.

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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Assay for apoptosis

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Apoptosis assays, based on

methodology, can be classified into six

major groups:

1. Cytomorphological alterations

2. DNA fragmentation

3. Detection of caspases, cleaved

substrates, regulators and inhibitors

4. Membrane alterations

5. Detection of apoptosis in whole mounts

6. Mitochondrial assays

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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

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DNA fragmentation occurs in the later phase of apoptosis.

TUNEL (Terminal dUTP Nick End-Labeling) assay quantifies

the incorporation of deoxyuridine triphosphate (dUTP) at single and

double stranded DNA breaks in a reaction catalyzed by the template

independent enzyme, terminal deoxynucleotidyl transferase (TdT).

Incorporated dUTP is labeled such that breaks can be quantified

either by flowcytometry, fluorescent microscopy, or light

microscopy.

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TUNEL positive cell

TUNEL negative cell

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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.

These cells can be demonstrated by bound with Fluorescein

isothyocyanate (FITC)-labeled Annexin V and detected with

fluorescent microscopy.

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.

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Apoptosis can also be visualized in whole mounts of embryos

or tissues using dyes such as acridine orange (AO), Nile blue

sulfate (NBS), and neutral red (NR).

Since these dyes are acidophilic, they are concentrated in areas

of high lysosomal and phagocytotic activity.

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Mitochondrial assays and cytochrome c release allow the

detection of changes in the early phase of the intrinsic pathway.

The mitochondrial outer membrane (MOM) collapses during

apoptosis, allowing detection with a fluorescent cationic dye.

Cytochrome c release from the mitochondria can also be assayed

using fluorescence and electron microscopy in living or fixed cells.

Apoptotic or anti-apoptotic regulator proteins such as Bax, Bid,

and Bcl-2 can also be detected using fluorescence and confocal

microscopy

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BCL2 inhibitors;

1)G3139 is an antisense oligodeoxynucleotide

targeting BCL2 mRNA resulting in RNAse H activation.

2)ABT263 is a small molecule mimetic of the BH3 domain of

the pro-apoptotic BAD protein that is currently in clinical trial in

chronic lymphatic leukaemia.

Caspaseinhibitors;

1) VX-765 is an orally active, reversible caspase-1 inhibitor that

was being developed for the treatment of inflammatory disorders.

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2) Emricasan is a novel, irreversible, orally active pan-caspase

inhibitor that has been investigated for the treatment of chronic

HCV infection and liver transplantation rejection.

3) NCX-1000, a small-molecule inhibitor that selectively inhibits

caspase-3, -8 and -9 in the micromolar range, was in phase II

clinical trials for the treatment of chronic liver disease.

4) PAC-1, a small molecule that induces both procaspase-3

activation in vitro and apoptosis in several cancer cell lines.

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با تشکر

Thanks for your attention

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