Transcript Slide 1

Anatomy and Human Biology 313-2005
Arun Dharmarajan
Professor
School of Anatomy & Human Biology
The University of Western Australia, Perth, Australia
Relevance of Apoptosis
in Health and Disease
• Apoptosis is a systematic and genetically
programmed process.
• Apoptosis is involved in tissue homeostasis and cell
differentiation.
• Apoptosis is directly involved in degenerative diseases,
autoimmune disorders, viral diseases, cancer, and
reproductive disorders.
Apoptosis Timeline
1842: Vogt recognised a form of
Physiological cell death.
1855: Flemming used the term “chromatolysis”
1951: Gluckmann described the morphological basis of
apoptosis.
1960: Lockshin-programmed cell death
1972: Wyllie and Kerr- Apoptosis
1985: Horvitz-apoptosis determined by several genes (The
Terminators)
The Good (blocks); The Bad (executes); The Ugly (activator of
apoptosis)
These genes are highly conserved throughtout evolution.
In Man, there are over 21 Goods (bcl-2 family), 14 Bads (the
caspases), BUT only one Ugly (Apaf-1).
APOPTOSIS
For every cell, there is a time to live and a time to die.
There are two ways in which cells die:
They are killed by injurious agents.
They are induced to commit suicide.
“Once we are in the land of the living, we will eventually die”
This is true not only for human beings but also for the cells
that make up our bodies
APOPTOSIS
Death by Injury
Cells that are damaged by injury, such as by
mechanical damage/ exposure to toxic chemicals
They (and their organelles like mitochondria) swell (because the ability
of the plasma membrane to control the passage of ions and water is
disrupted). The cell contents leak out, leading to inflammation of
surrounding tissues.
Apoptosis
How to die….
• A discrete, active and energy dependent
form of cell death. Executed roughly 20
times as rapidly as mitosis.
• Several characteristic morphological
features;
Chromatin condensation, Membrane blebbing,
DNA fragmentation (180bp multiples), No inflammatory
response.
APOPTOSIS
Death by suicide
Cells that are induced to commit suicide:
shrink;
develop bubble-like blebs on their surface;
have the chromatin (DNA and protein) in their nucleus degraded;
have their mitochondria break down with the release of
cytochrome c; break into small, membrane-wrapped, fragments.
The phospholipid phosphatidylserine, which is normally hidden
within the plasma membrane, is exposed on the surface.
This is bound by receptors on phagocytic cells like macrophages
and dendritic cells which then engulf the cell fragments.
The phagocytic cells secrete cytokines that inhibit inflammation
(e.g., IL-10 and TGF-β)
APOPTOSIS
APOPTOSIS
Why should a cell commit suicide?
There are two different reasons.
1. Programmed cell death is as needed for proper
development as mitosis is.
2. Programmed cell death is needed to destroy cells
that represent a threat to the integrity of the
organism. e.g
Cells infected with viruses
Cells of the immune system
Cells with DNA damage
Cancer cells
APOPTOSIS
What makes a cell decide to commit suicide?
The balance between:
•the withdrawal of positive signals; that is, signals needed
for continued survival e.g Growth Factors/Hormones
•the receipt of negative signals e.g. UV/oxidants/
X-Rays/Chemo
Death activators: TNF/ Fas/Lymphotoxin
APOPTOSIS
The Mechanisms of Apoptosis
There are 3 different mechanisms by which a cell commits suicide by apoptosis.
One generated by signals arising within the cell;
another triggered by death activators binding to receptors at the cell surface:
TNF-α
Lymphotoxin
Fas ligand (FasL)
A third that may be triggered by dangerous reactive oxygen species.
APOPTOSIS
1. Apoptosis triggered by internal signals: the intrinsic or mitochondrial
pathway
In a healthy cell, the outer membranes of its mitochondria express the protein
Bcl-2 on their surface. Bcl-2 is bound to a molecule of the protein Apaf-1
("apoptotic protease activating factor-1".
Internal damage to the cell (e.g., from reactive oxygen species) causes
Bcl-2 to release Apaf-1; a related protein, Bax, to penetrate mitochondrial
membranes, causing cytochrome c to leak out.
The released cytochrome c and Apaf-1 bind to molecules of caspase 9.
The resulting complex of cytochrome c/Apaf-1 /caspase 9 /(and ATP)
is called the apoptosome.
APOPTOSIS
2. Apoptosis triggered by external signals:
the extrinsic or death receptor pathway
Fas and the TNF receptor are integral membrane proteins with their
receptor domains exposed at the surface of the cell
binding of the complementary death activator (FasL and TNF
respectively) transmits a signal to the cytoplasm that leads to activation of
caspase 8 caspase 8 (like caspase 9) initiates a cascade of caspase
activation leading to phagocytosis of the cell.
APOPTOSIS
Apoptosis-Inducing Factor (AIF)
Neurons, and perhaps other cells, have another way to self-destruct that —
unlike the two paths described above — does not use caspases.
Apoptosis-inducing factor (AIF) is a protein that is normally located in the
intermembrane space of mitochondria.
When the cell receives a signal telling it that it is time to die, AIF
is released from the mitochondria (like the release of cytochrome c in the
first pathway); migrates into the nucleus; binds to DNA, which triggers the
destruction of the DNA and cell death.
APOPTOSIS
Apoptosis and Cancer
Some cancer-causing viruses use tricks to prevent apoptosis of the cells they
have transformed.
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 a cause of
Burkitt's lymphoma 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 the cancer cell to continue to proliferate).
APOPTOSIS
Even cancer cells produced without the participation of viruses may have
tricks to avoid apoptosis.
Some B-cell leukemias and lymphomas express high levels of Bcl-2,
thus blocking apoptotic signals they may receive.
Melanoma (the most dangerous type of skin cancer) cells avoid
apoptosis by inhibiting the expression of the gene encoding Apaf-1.
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
by the mechanism shown above.
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).
APOPTOSIS
The Mechanisms of Apoptosis
There are 3 different mechanisms by which a cell commits suicide by apoptosis.
One generated by signals arising within the cell;
another triggered by death activators binding to receptors at the cell surface:
TNF-α
Lymphotoxin
Fas ligand (FasL)
A third that may be triggered by dangerous reactive oxygen species.
TNF Signal Transduction
TNFR1
TNFR2
JNK
CERAMIDE
SPHINGOSINE
NITRIC OXIDE
P38 MAPK
NF-B
ERK 1&2
CASPASES
CLEAVAGE OF DEATH
SUBSTRATES
APOPTOTIC PHENOTYPE
CELL SURVIVAL
Low MW DNA labelling
(Fold change vs. Time 0)
Apoptosis in human placenta
9
8
7
6
5
4
3
2
1
0
*
Time 0
T0 C4 S4
Control 4
Sod 4
APOPTOSIS
Assays that Measure DNA Fragmentation (HMW DNA, DNA
Content)
Assays that Examine Chromatin Morphology
Assays that Measure DNA Strand Breaks (Nicks) and DNA
Fragmentation (Staggered DNA Ends)
Assays that Detect Phosphatidylserine on the Surface of
Apoptotic Cells
Assays that Measure Plasma Membrane Damage/Leakage
Comet Assay
SPONTANEOUS APOPTOSIS
DNA labelling following spontaneous apoptosis
0h 2h 4h 6h
Fold changes vs. CONT 0h
0h
1x
2h
6x
4h
8x
6h
12x
Slide 54
METHOD
• Cultured cells or frozen section
• Embed cells/section in agarose
• Lyse cell membrane
• Equilibrate in electrophoresis buffer
• Electrophoresis
•Stain and view
Comet Assay
•Visual measure of DNA damage
•Cells lysed, electrophoresed, stained
•Image produced resemble comet
Advantages
1. Distinguish necrosis from apoptosis
2. Identify cell types undergoing apoptosis