MECHANISMS OF DISEASE II: CELL DAMAGE AND CELL DEATH

Question 1

What is the function of necrosis?

Answer 1

Removes damaged cells from an organism Failure to do so may lead to chronic inflammation Necrosis causes acute inflammation(to prevent further bigger inflammation) to clear cell debris via phagocytosis

Question 2

What are the causes of necrosis?

Answer 2

Usually lack of blood supply, e.g. injury, infection, cancer, infarction, inflammation (As the distance away from the blood vessel increases, the pH drops and partial pressure of oxygen drops significantly also- step by step explanation of the effect of this on flashcard 3 below)

Question 3

What are the steps of necrosis?

Answer 3

1. Result of an injurious agent or event.(Whole groups of cells are affected.) 2. Initial events are reversible, later ones are not. 3. Lack of oxygen prevents ATP production. 4. Cells swell due to influx of water (ATP is required for ion pumps to work).(due to osmosis here) 5. Lysosomes rupture; enzymes degrade other organelles and nuclear material hapzardly 6. Cellular debris released, triggering inflammation

Question 4

What are some nuclear changes that occur in a cell during necrosis? (from a microscope view)

Answer 4

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

Question 5

What are some cytoplasmic changes that occur in necrosis of a cell? (microscopic view)

Answer 5

1. Opacification:(cytoplasm becomes more white instead of a see-through watery colour) protein denaturation & aggregation. 2. Complete digestion of cells by enzymes causing cell to liquify (liquefactive necrosis).

Question 6

What are some biochemical changes that occur in necrosis of a cell?

Answer 6

1. Release of enzymes such as creatine kinase or lactate dehydrogenase 2. Release of other proteins such as myoglobin These biochemical changes are useful in the clinic to measure the extent of tissue damage!

Question 7

What is apoptosis and what is it involved in?

Answer 7

Selective process for the deletion of superfluous, infected or transformed cells. Involved in:- Embryogenesis Metamorphosis Normal tissue turnover Endocrine-dependent tissue atrophy A variety of pathological conditions

Question 8

What happens in apoptosis?

Answer 8

1. Programmed cell death of one or a few cells. 2. Events are irreversible and energy (ATP) dependent. 3. Cells shrink as the cytoskeleton is disassembled. 4. Orderly packaging of organelles and nuclear fragments into membrane bound vesicles. 5. New molecules are expressed on vesicle membranes that stimulate phagocytosis without an inflammatory response (so a v clean way of disposing cellular content)

Question 9

What is a distinct feature in regards to number of cells involved in necrosis and apoptosis? What about in regards to reversibility?

Answer 9

Necrosis- multiple cells at once Apoptosis- v selective, usually one or so cells at a time Necrosis- not every part/stage is irreversible Apoptosis -ALL events are irreversible and steps require ATP

Question 10

What cytoplasmic changes can be seen when cell apoptosis happens?

Answer 10

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.

Question 11

What nuclear changes can be seen when cell apoptosis happens?

Answer 11

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

Question 12

What biochemical changes can be seen when cell apoptosis happens?

Answer 12

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 13

What are some examples of things that cause apoptosis?

Answer 13

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 to 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 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 14

What are caspases?

Answer 14

a family of proteases whose activation is central to all types of apoptosis Caspases are the point of convergence for causes of apoptosis, e.g. Extrinsic causes intrinsic causes . . . . . . Caspases………………………….> apoptosis Caspases are cysteine proteases (cysteine aspartate-specific proteases) Caspases form an activation cascade, where one cleaves and activates the next (analogous to kinase cascades) (initiator caspases and effector caspases)

Question 15

What are the 2 types of apoptosis?

Answer 15

Intrinsic: DNA damage – p53-dependent pathway Interruption of the cell cycle Inhibition of protein synthesis Viral Infection- ie once virus is in the cell Change in redox state Extrinsic: (relative to the cell not the body) Withdrawal of survival factors e.g. mitogens Extracellular signals (e.g. TNF) T cell or NK (Natural Killer) (e.g. Granzyme).

Question 16

What does caspase activation lead to?

Answer 16

Caspase activation leads to characteristic morphological changes, such as shrinkage, chromatin condensation, DNA fragmentation and plasma membrane blebbing. Initiator caspases activate themselves when in close proximity Activation, therefore, means bringing initiator caspases together

Question 17

What is extrinsic apoptosis induced by?

Question 18

What is intrinsic apoptosis induced by?

Question 19

How is the release of cytochrome c from the mitochondria regulated?

Question 20

IF BCL-2 FAMILY PROTEINS REGULATE CYTOCHROME C RELEASE FROM mitochondria, WHAT regulates BCL-2 PROTEINS?

Question 21

By what 3 mechanisms is cell death caused by?

Answer 21

• 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 22

Describe the process of necrosis

Answer 22

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 23

What can cause necrosis?

Answer 23

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 24

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

Answer 24

• 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 25

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

Answer 25

Nuclear Changes
1. Chromatin condensation/shrinkage.

2. Fragmentation of nucleus.
3. Dissolution of the chromatin by DNAse.
   Cytoplasmic Changes
4. Opacification: denaturation of proteins with aggregation  The tissue turns dark
5. Complete digestion of cells by enzymes causing cell to liquify (liquefactive necrosis).
   Biochemical Changes
6. Release of enzymes such as creatine kinase or lactate dehydrogenase
7. Release of proteins such as myoglobin
   • These biochemical changes are useful in the clinic to measure the extent of tissue damage.

Question 26

What is Astrocytoma?

Answer 26

• 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 27

Compare a normal and necrotic kidney

Answer 27

• 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 28

What are the 2 functions of necrosis?

Answer 28

• Removes damaged cells from an organism

* Failure to do so may lead to chronic inflammation.

Question 29

How is necrosis a reversible process?

Answer 29

• 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 30

What are the 7 principles of apoptosis?

Answer 30

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 31

What are the functions of apoptosis?

Answer 31

• 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 32

Describe the process of apoptosis induced by deprivation of growth factor

Answer 32

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 33

What factors promote life and death

Question 34

What are the 2 types of apoptosis?

Question 35

What are Caspases?

Answer 35

• 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 36

How is Apoptosis is mediated by an intracellular proteolytic cascade

Answer 36

• 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 37

What does the caspase cascade lead to?

Answer 37

• 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 38

What are the morphological features of apoptosis

Answer 38

• 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 39

What is DNA fragmentation?

Answer 39

• 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.