Cell Damage and Cell Death

Question 1

List some causes and mechanisms of cell damage/death.

Answer 1

GENETIC:

• Abnormal chromosomes (deletions/translocatioNs)
• Increased fragility (Fanconi’s anaemia)
• Failure of repair (Xeroderma pigmentosa)
• Inborn errors (Storage disorders ie. Tay Sachs disease)

INFLAMMATION:

• Trauma
• Thrombo-embolism
• Atherosclerosis
• Vasculitis

PHYSICAL:

• Irradiation
• Heat
• Cold
• Barotrauma

TRAUMATIC DAMAGE:

• Interruption of blood supply
• Direct rupture of cells
• Entry of foreign agents

INFECTION:

• Toxic agents
• Competition for nutrients
• Intracellular replication (viruses/mycobacteria provoking an immune response)

CHEMICAL:

• Acids/corrosives
• Specific actions e.g. enzymes
• Interference with metabolism e.g. alcohol

Question 2

What are the three basic mechanisms of cell death?

Answer 2

1) Necrosis
2) Apoptosis
3) Autophagic cell death

Question 3

Expand on necrosis as a cell death mechanism.

Answer 3

It is the most common cause of cell death.

It occurs after stresses, such as ischeamia, trauma, chemical injury, etc.

Question 4

Expand on apoptosis as a cell death mechanism.

Answer 4

It is programmed cell death designed to eliminate unwanted host cells through activation of a co-ordinated, internally programmed series of events effected by a dedicated set of gene products.

Question 5

Expand on autophagic cell death as a cell death mechanism.

Answer 5

Autophagy is responsible for the degradation of normal proteins involved in cellular remodeling found during metamorphosis, aging and differentiation.

It is also for the digestion and removal of abnormal proteins that would otherwise accumulate following toxin exposure, cancer, or disease.

An example is the death of breast cancer cells induced by Tamoxifen.

Question 6

What are some causes of necrosis?

Answer 6

It’s usually caused by:

• lack of blood supply to cells or tissues
• injury
• infection
• cancer
• infarction
• inflammation

Question 7

What are the steps to necrosis?

Answer 7

1. Whole groups of cells are affected.
2. It’s as a result of an injurious agent or event.
3. Reversible events proceed irreversibly.
4. Energy deprivation causes changes (e.g. cells unable to produce ATP because of oxygen deprivation).
5. Cells swell due to influx of water (ATP is required for ion pumps to work).
6. Haphazard destruction of organelles and nuclear material by enzymes from ruptured lysosomes.
7. Cellular debris stimulates an inflammatory cell response.

Question 8

What are the different microscopic changes in necrosis?

Answer 8

NUCLEAR CHANGES:

1. Chromatin condensation/shrinkage.
2. Fragmentation of nucleus.
3. Dissolution of the chromatin by DNAse.

CYTOPLASMIC CHANGES:

1. Opacification: denaturation of proteins with aggregation.
2. Complete digestion of cells by enzymes causing cell to liquify (liquefactive necrosis).

BIOCHEMICAL CHANGES:

1. Release of enzymes such as creatine kinase or lactate dehydrogenase
2. Release of proteins such as myoglobin

These biochemical changes are useful in the clinic to measure the extent of tissue damage!

Question 9

What is the function of necrosis?

Answer 9

It removes damaged cells from an organism.

Failure to do so may lead to chronic inflammation.

Question 10

List some functions of apoptosis.

Answer 10

Apoptosis is a selective process for the deletion of superfluous, infected or transformed cells.

It’s involved in:-

• Embryogenesis
• Metamorphosis
• Normal tissue turnover
• Endocrine-dependent tissue atrophy
• A variety of pathological conditions

Question 11

List some examples of apoptosis.

Answer 11

1. Cell death in embryonic hand to form individual fingers.
2. Apoptosis induced by growth factor deprivation (neuronal death from lack of NGF).
3. DNA damage-mediated apoptosis. If DNA is damaged due to radiation or chemo therapeutic agents, p53 (tumour suppressor gene product) accumulates. This arrests the cell cycle enabling the cell repair the damage. If repair process fails, p53 triggers apoptosis.
4. Cell death in tumours causing regression.
5. Cell death in viral diseases (ie viral hepatitis).
6. Cell death induced by cytotoxic T cells (ie. Cellular immune rejection or graft vs. host disease).
7. Death of neutrophils during an acute inflammatory response.
8. Death of immune cells (both T and B lymphocytes) after depletion of cytokines as well of death of autoreactive T cells in the developing thymus.

Question 12

List some factors influencing the balance of life and death at the cellular level.

Answer 12

For SURVIVAL:

• cell-cell and/or cell-matrix contacts
• growth factors
• cytokines

For APOPTOSIS:

• disruption of cell-cell and/or cell-matrix contacts
• lack of growth factors
• death domain ligands
• DNA damaging agents

Question 13

Describe the two types of apoptosis.

Answer 13

INTRINSIC:

• DNA damage – p53-dependent pathway
• Interruption of the cell cycle
• Inhibition of protein synthesis
• Viral Infection
• Change in redox state

EXTRINSIC:

• Withdrawal of growth factors (e.g. IL-3)
• Extracellular signals (e.g. TNF)
• T cell or NK (Natural Killer) (e.g. Granzyme).

Question 14

Describe caspases (cysteine aspartate-specific proteases).

Answer 14

Caspases are cysteine proteases that play a central role in the initiation of apoptosis.

Most proteases are synthesised as inactive precursors requiring activation (usually partial digestion by another protease).

Question 15

Apoptosis is mediated by an intracellular proteolytic cascade.

Expand.

Answer 15

First we have to have caspase activation. A inactive caspase Y is activated by an activated caspase X. X cleaves the domain off, which deems it active.

With caspase X, it can activate many caspase Y’s, which go on to activate even more molecules of caspase Z. Thus, we have an increased number of effector caspases, known as the caspase cascade.

Question 16

What changes does caspase activation cause in the cell?

Answer 16

Caspase activation leads to characteristic morphological changes of the cell such as shrinkage, chromatin condensation, DNA fragmentation and plasma membrane blebbing.

Question 17

What are some ways to visualise cell death?

Answer 17

You could use transmission electron micrography or scanning electron micrography.

Question 18

How can you differentiate different kinds of cell death based on DNA fargmentation?

Answer 18

You would take a sample of purified DNA from the cell in question and run it through gel electrophoresis.

Cells that are normal will have their DNA at one specific band. Cells that are apoptosing will have multiple bands of decreasing levels at certain intervals, while cells that are necrosing will have continuously decreasing DNA.

Question 19

Describe the process of apoptosis.

Answer 19

1. One single or a few cells are selected.
2. They undergo programmed cell death.
3. It’s irreversible once initiated.
4. The events are energy driven.
5. Cells shrink as the cytoskeleton is disassembled.
6. The orderly packaging of organelles and nuclear fragments in membrane bound vesicles occurs.
7. New molecules expressed on vesicle membranes stimulate phagocytosis, so there is no inflammatory response.

Question 20

What are the different microscopic changes in apoptosis?

Answer 20

NUCLEAR CHANGES:

1. Nuclear chromatin condenses on nuclear membrane.
2. DNA cleavage.

CYTOPLASMIC CHANGES:

1. Shrinkage of cell. Organelles packaged into membrane vesicles.
2. Cell fragmentation. Membrane bound vesicles bud off.
3. Phagocytosis of cell fragments by macrophage and adjacent cell.
4. No leakage of cytosolic components.

BIOCHEMICAL CHANGES:

1. Expression of charged sugar molecules on outer surface of cell membranes (recognised by macrophages to enhance phagocytosis)
2. Protein cleavage by proteases, caspases.

Question 21

How do we activate the initiator caspases?

Answer 21

We do so by induced proximity.

For example:

• In response to receptor dimerization upon ligand binding or
• Cytochrome C release from the mitochondria.

Question 22

With ligand-induce dimerisation, what are the key players?

Answer 22

In the extrinsic pathway, we have:

• the RECEPTOR, with a ligand binding domain and a death domain
• the DEATH ADAPTOR, with a death domain and a death effector domain
• the PROCASPASE-8, with a death effector domain and a protease domain

When they all bind together, the PROCASPASE-8’s will all be in close proximity, which will cause their autoproteolysis, activating them.

Question 23

Describe cytochrome C.

Answer 23

It’s a mitochondrial matrix protein.

It’s been known for many years to be released in response to oxidative stress by a “permeability transition”.

Any inducers of the permeability transition also eventually induce apoptosis.

Question 24

What are the key players in cytochrome-C induced apoptosis?

Answer 24

In the intrinsic pathway, the key players are:

• cytochrome C
• APAF-1, with a cytochrome-C binding site, APAF domain, and a caspase recruitment domain (CARD)
• PROCASPASE-9, with a caspase recruitment domain (CARD) and a protease domain

Again, when the key players come together, the PROCASPASE-9’s end up in close proximity to each other, causing autoproteolysis, and their activation.

Question 25

How is the release of cytochrome C regulated?

Answer 25

Bcl-2 is a member of a multi-gene family in mammals.

There are two types of units:
ANTI-APOPTOTIC:
- bcl-2
- bcl-XL

PRO-APOPTOTIC:

• Bax
• Bad
• Bid

The bcl-2 family members form dimers.
If one or more of the dimer units is anti-apoptotic, they promote life. If both are pro-apoptotic, then they promote death.

Bax proteins can congregate to form a channel in the mitochondrial membrane, which would allow the cytochrome-C out. With the anti-apoptotic bcl-2 units, they block that channel to avoid its release, but the pro-apoptotic ones dimerise and remove them, allowing its release.

Question 26

How does p53 relate to apoptosis?

Answer 26

Mutations in the p53 gene are the most common mutations

in cancer. Some mutations destroy the ability of p53 to induce apoptosis.