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).

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

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.

ApoptosisHow 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 suicideCells 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

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/ChemoDeath activators: TNF/ Fas/Lymphotoxin

APOPTOSIS

The Mechanisms of ApoptosisThere 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 pathwayIn 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

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).

The Mechanisms of ApoptosisThere 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

TNF Signal Transduction

CERAMIDE

P38 MAPK

CLEAVAGE OF DEATHSUBSTRATES

APOPTOTIC PHENOTYPE

TNFR2

TNFR1

ERK 1&2SPHINGOSINE

CELL SURVIVAL

CASPASES

JNK

NF-B

NITRIC OXIDE

Apoptosis in human placenta

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Assays that Measure DNA Fragmentation (HMW DNA, DNA Content)Assays that Examine Chromatin MorphologyAssays that Measure DNA Strand Breaks (Nicks) and DNA Fragmentation (Staggered DNA Ends)Assays that Detect Phosphatidylserine on the Surface of Apoptotic CellsAssays that Measure Plasma Membrane Damage/LeakageComet Assay

APOPTOSIS

SPONTANEOUS APOPTOSISDNA labelling following spontaneous apoptosis

0h 2h 4h 6h Fold changes vs. CONT 0h

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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