Molecular Human Reproduction-2005

Apoptosis and Reproduction

Arun Dharmarajan

School of Anatomy amp Human Biology

The University of Western Australia Perth Australia

Relevance of Apoptosis in Health and Disease

bull Apoptosis is a systematic and genetically programmed process

bull Apoptosis is involved in tissue homeostasis and cell differentiation

bull 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 ldquochromatolysisrdquo1951 Gluckmann described the morphological basis of

apoptosis1960 Lockshin-programmed cell death1972 Wyllie and Kerr- Apoptosis1985 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 evolutionIn 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

ldquoOnce we are in the land of the living we will eventually dierdquo This is true not only for human beings but also for the cells

that make up our bodies

APOPTOSIS

APOPTOSIS

Death by InjuryCells 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

Relevance of Apoptosis in Health and Disease

bull Apoptosis is a systematic and genetically programmed process

bull Apoptosis is involved in tissue homeostasis and cell differentiation

bull 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 ldquochromatolysisrdquo1951 Gluckmann described the morphological basis of

apoptosis1960 Lockshin-programmed cell death1972 Wyllie and Kerr- Apoptosis1985 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 evolutionIn 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

ldquoOnce we are in the land of the living we will eventually dierdquo This is true not only for human beings but also for the cells

that make up our bodies

APOPTOSIS

APOPTOSIS

Death by InjuryCells 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 Timeline

1842 Vogt recognised a form of Physiological cell death

1855 Flemming used the term ldquochromatolysisrdquo1951 Gluckmann described the morphological basis of

apoptosis1960 Lockshin-programmed cell death1972 Wyllie and Kerr- Apoptosis1985 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 evolutionIn 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

ldquoOnce we are in the land of the living we will eventually dierdquo This is true not only for human beings but also for the cells

that make up our bodies

APOPTOSIS

APOPTOSIS

Death by InjuryCells 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

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

ldquoOnce we are in the land of the living we will eventually dierdquo This is true not only for human beings but also for the cells

that make up our bodies

APOPTOSIS

APOPTOSIS

Death by InjuryCells 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

Death by InjuryCells 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 diehellip

bull A discrete active and energy dependent form of cell death Executed roughly 20 times as rapidly as mitosis

bull Several characteristic morphological features Chromatin condensation Membrane blebbing DNA fragmentation (180bp multiples) No inflammatory response

APOPTOSISDeath 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 macrophagesand dendritic cells which then engulf the cell fragments The phagocytic cells secrete cytokines that inhibit inflammation(eg IL-10 and TGF-β)

APOPTOSIS

APOPTOSISDeath 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 macrophagesand dendritic cells which then engulf the cell fragments The phagocytic cells secrete cytokines that inhibit inflammation(eg IL-10 and TGF-β)

APOPTOSIS

APOPTOSISWhy should a cell commit suicideThere 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 eg

Cells infected with virusesCells of the immune systemCells with DNA damageCancer cells

What makes a cell decide to commit suicide

The balance between

bullthe withdrawal of positive signals that is signals needed for continued survival eg Growth FactorsHormones

bullthe receipt of negative signals eg UVoxidantsX-RaysChemoDeath activators TNF FasLymphotoxin

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 LymphotoxinFas ligand (FasL)

A third that may be triggered by dangerous reactive oxygen species

APOPTOSIS

What makes a cell decide to commit suicide

The balance between

bullthe withdrawal of positive signals that is signals needed for continued survival eg Growth FactorsHormones

bullthe receipt of negative signals eg UVoxidantsX-RaysChemoDeath activators TNF FasLymphotoxin

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 LymphotoxinFas ligand (FasL)

A third that may be triggered by dangerous reactive oxygen species

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 LymphotoxinFas 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 (eg 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 cApaf-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 TNFrespectively) transmits a signal to the cytoplasm that leads to activation of caspase 8 caspase 8 (like caspase 9) initiates a cascade of caspaseactivation leading to phagocytosis of the cell

APOPTOSIS

Apoptosis-Inducing Factor (AIF)

Neurons and perhaps other cells have another way to self-destruct that mdashunlike the two paths described above mdash does not use caspases Apoptosis-inducing factor (AIF) is a protein that is normally located in theintermembrane 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 Burkitts 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

APOPTOSISEven 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 LymphotoxinFas ligand (FasL)

A third that may be triggered by dangerous reactive oxygen species

APOPTOSIS

TNFα Signal Transduction

CERAMIDE

P38 MAPK

CLEAVAGE OF DEATHSUBSTRATES

APOPTOTIC PHENOTYPE

TNFR2

TNFR1

ERK 1amp2SPHINGOSINE

CELL SURVIVAL

CASPASES

JNK

NF-κB

NITRIC OXIDE

Apoptosis in human placenta

0123456789

Time 0 Control 4 Sod 4

Low

MW

DNA

labe

lling

(F

old

chan

ge v

s T

ime

0)

T0 C4 S4

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 DamageLeakageComet Assay

APOPTOSIS

Apoptosis in human placenta

0123456789

Time 0 Control 4 Sod 4

Low

MW

DNA

labe

lling

(F

old

chan

ge v

s T

ime

0)

T0 C4 S4

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 DamageLeakageComet Assay

APOPTOSIS

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 DamageLeakageComet Assay

APOPTOSIS

SPONTANEOUS APOPTOSISDNA labelling following spontaneous apoptosis

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

0h 1x

2h

4h

6h

8x

6x

12x

Slide 54

Slide 54