Mechanisms of Disease II: Cell Damage and Cell Death

Question 1

By what 3 mechanisms is cell death caused by?

Answer 1

• Necrosis: Most common cause of cell death. Occurs after stresses such as ischemia (lack of O2) , trauma, chemical injury. ‘Death by accident’.
• Apoptosis: 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. ‘Death by design’.
• Autophagic cell death: Autophagy is responsible for the degradation of normal proteins involved in cellular remodelling found during metamorphosis, aging and differentiation as well as 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 2

Describe the process of necrosis

Answer 2

1. Whole groups of cells are affected.
2. Result of an injurious agent or event.
3. Reversible events proceed irreversible.
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. Affects near by healthy cells, sugars, proteins etc.
8. Cellular debris stimulates an inflammatory cell response

Question 3

What can cause necrosis?

Answer 3

Usually caused by lack of blood supply (so no ATP or O2) to cells or tissues, e.g.
• Injury (Car crash)
• Infection (Competition for nutrients involved)
• Cancer
• (Cancer can lead to necrosis  as the cells expand, it compresses neighbouring blood vessels  restriction of blood flow)
• Infarction
• Inflammation (Tissues expand so this restricts blood vessels)

Question 4

What is the relationship between distance along blood vessel and pH and pO2

Answer 4

• It shows that pH and oxygen levels are both very high when you are closer to the blood vessels. They both decrease quite rapidly.
• As you move along a blood vessel pH and pO2 decrease.

Question 5

What are the nuclear changes, cytoplasmic changes and biochemical changes of necrosis?

Answer 5

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  The tissue turns dark
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 6

What is Astrocytoma?

Answer 6

• Tumour is erasing the normal histology shape or brain cells.
• An example of necrotic tissue
• Astrocytoma count for 60% of brain tumours
• It’s a cancerous tissue
• The cancer cells are erasing nearby tissue  the nearby tissues undergo necrosis  the necrotic tissue is darker.

Question 7

Compare a normal and necrotic kidney

Answer 7

• Glomurli, E and T staining in the kidney. Clearly see cell DNA nucleus.
• What we can see in the necrotic glomeruli the DNA is totally degraded so lack of DNA staining. Known as ghost cells they were there but nothing inside the cell compartment.
• An example of necrotic glomeruli
• The DNA is totally degraded
• Called ghost cells  it looks like the cell is there but if you look closely, there is nothing inside

Question 8

What are the 2 functions of necrosis?

Answer 8

• Removes damaged cells from an organism

* Failure to do so may lead to chronic inflammation.

Question 9

How is necrosis a reversible process?

Answer 9

• The cells do not receive a blood supply and so do not receive oxygen.
o The lack of oxygen means they are unable to generate ATP via oxidative phosphorylation.
• The ion pumps in the cell membrane are one of the first molecules to stop working with a lack of ATP.
o If the ion pumps don’t work, the water balance is not regulated and so the cells start to swell  because water starts coming into the cells.
• We can restore the function by providing cells with ATP.
o Therefore, necrosis is a reversible process.
• However, if the amount of water entering the cell is massive, you reach the point of no return  the swelling is irreversible.
o The organelles within, such as the nucleus, mitochondria and lysosomes swell as well.
• Eventually the fate of the cell is to disintegrate.
o The cells then release all their intra cellular components.
o All the organelles explode which can lead to the release of all the enzymes contained in lysosomes (e.g. proteases, lipases, glucosidases)

Question 10

What are the 7 principles of apoptosis?

Answer 10

1. Single or few cells selected.
2. Programmed cell death.
3. Irreversible once initiated.
4. Events are energy driven.
5. Cells shrink as the cytoskeleton is disassembled.
6. Orderly packaging of organelles and nuclear fragments in membrane bound vesicles.
7. New molecules expressed on vesicle membranes stimulate phagocytosis, no inflammatory response.

Question 11

What are the functions of apoptosis?

Answer 11

• Selective process for the deletion of superfluous (not required by organism anymore), infected or transformed cells. Involved in:
• Metamorphosis
• Normal tissue turnover
• Endocrine-dependent tissue atrophy
• A variety of pathological conditions
• Embryogenesis

Question 12

Describe the process of apoptosis induced by deprivation of growth factor

Answer 12

1. Apoptosis induced by growth factor deprivation (neuronal death from lack of NGF).
2. 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.
3. Cell death in tumours causing regression.
4. Cell death in viral diseases (i.e. viral hepatitis).
5. Cell death induced by cytotoxic T cells (i.e. Cellular immune rejection or graft vs. host disease).
6. Death of neutrophils during an acute inflammatory response.
7. 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 13

What factors promote life and death

Answer 13

On image

Question 14

What are the 2 types of apoptosis?

Answer 14

On image

Question 15

What are Caspases?

Answer 15

• 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).
• Present in cells as synth as precursors

Question 16

How is Apoptosis is mediated by an intracellular proteolytic cascade

Answer 16

• Apoptosis is mediated by an intracellular proteolytic cascade
• Procaspase Y is inactive but is activated and cleaved by active caspase X.
• This cleaves specifically to form a large and small subunit which make a dimer.
• Now you have active caspase Y.
• Caspase X is also initially expressed as an inactive form in cells.

Question 17

What does the caspase cascade lead to?

Answer 17

• Caspase activation leads to characteristic morphological changes of the cell such as shrinkage, chromatin condensation, DNA fragmentation and plasma membrane blebbing.
• When you trigger apoptosis, you have an active initiator caspase (usually 8/9).
• The active form of this caspase will activate other caspases and as a result you start to cleave cytosolic proteins that contain cysteine-aspartate residues.
• The actin cytoskeleton starts to breakdown and the cell starts to collapse.
• This caspase can then activate many effector caspases (1,3,6,7) which leads to a massive amplification of proteolysis. Eventually, you also end up cleaving the nuclear lamin (this is a protein which is required for the nuclear envelope).

Question 18

What are the morphological features of apoptosis

Answer 18

• Caspase activation leads to characteristic morphological changes of the cell such as shrinkage, chromatin condensation, DNA fragmentation and plasma membrane blebbing.
• This is what cells looks like under the microscope.
• The healthy cells will receive a stimulus that triggers apoptosis.
• The cells become rounded and start to collapse mainly due to the actin cytoskeleton being degraded.
• If you follow the cells over time, they would detach from the substrate and would be seen floating.
• Start to see the formation of blebs.
• Inside blebs, there are mitochondria, lysosomes, etc.
• Eventually, these vesicles bud off the cells and are coated with sugars.
• This is a signal for them to be engulfed by nearby macrophages via phagocytosis.

Question 19

What is DNA fragmentation?

Answer 19

• Healthy cells and purify genomic DNA.
• On right genomic cells, apoptosis cells and necrosis cells. Necrosis cells release a lot of enzymes so the nucleosomes particles.
• DNA fragmentation uses electrophoresis.
• You can see if your cells are dying via necrosis or apoptosis.
• You purify genomic DNA from cells and run it on an agarose gel.
• The genomic DNA of a normal cell has a high molecular weight, so it does not run on the gel.
• Cell undergoing apoptosis show a very clear ladder structure.
• Each one of the fragments signifies 150 base pairs wrapped around the nucleosome.
• In apoptotic cells, the nucleosomes are intact as well as the DNA surrounding them.
• In cells undergoing necrosis, the fragmentation of DNA is random.
• This is because the cells don’t have nucleosomes.
• In necrotic cells, the enzymes that are released by the cell cleave all the nucleosomes and so the DNA is naked and broken randomly.