Cell Damage and Cell Death

Question 1

What are the mechanisms by which cell damage or cell death can occur?

Answer 1

“1. Genetic Factors.

2. Inflammation.
3. Physical Damage.
4. Traumatic Damage.
5. Infection.
6. Chemical.”

Question 2

What genetic factors can result in cell damage or cell death?

Answer 2

”- Abnormal number of chromosomes:
Aneuploidy.

• Abnormal chromosomes.
  Deletions/translocations.
• Increased fragility.
  Fanconi’s Anaemia.
• Failure of repair.
  Xeroderma Pigmentosa.
• Inborn errors.
  Storage disorders ie. Tay Sachs Disease.”

Question 3

How can inflammation result in cell damage or cell death?

Answer 3

“Trauma.

Thrombo-embolism.

Atherosclerosis.

Vasculitis.”

Question 4

What forms of physical damage can cause cell death or damage?

Answer 4

“Irradiation.

Heat.

Cold.

Barotrauma.”

Question 5

What forms of traumatic damage can cause cell death or damage?

Answer 5

“Interruption of blood supply.

Direct rupture of cells.

Entry of foreign agents.”

Question 6

How can infection cause cell death or damage?

Answer 6

“Toxic agents.

Competition for nutrients.

Intracellular replication.

Viruses/mycobacteria provoking an immune response.”

Question 7

How can chemicals cause cell death or cell damage?

Answer 7

“Acids/corrosives.

Specific actions e.g. enzymes,

Interference with metabolism e.g. alcohol.”

Question 8

What are the three basic mechanisms that cause Cell Death?

Answer 8

“1. Necrosis.

2. Apoptosis.
3. Autophagic Cell Death.”

Question 9

What is Necrosis and when does it occur?

Answer 9

“Most Common cause of Cell Death.

• Occurs after stresses such as ischemia, trauma, chemical injury.
• Accidental death.
• Whole groups of cells are affected.
• Reversible events proceed to become irreversible.”

Question 10

What is Apoptosis and what is its purpose?

Answer 10

“Programmed Cell Death:

• Designed to eliminate unwanted host cells.
• This is done through the activation of a co-ordinated, internally programmed series of events.
• Which are effected by a dedicated set of gene products.
• Therefore this is death by design.
• These cells have finished the job they were supposed to do.”

Question 11

What is Autophagic Cell Death?

Answer 11

“Responsible for the Degradation of Normal Proteins:

• Involved in cellular remodeling.
• Which is during metamorphosis, ageing and differentiation.
• Allows the digestion and removal of abnormal proteins that would otherwise accumulate after toxin exposure, cancer, or disease.
• An example is the death of breast cancer cells induced by Tamoxifen.
• Allows cell to get rid of organelles that are ageing.”

Question 12

List the causes of Necrosis?

Answer 12

“1. Lack of blood supply: To cells or tissues, e.g.injury.

2. Infection: Excessive growth of pathogens steal the O2 supply of the body.
3. Cancer: Press against blood vessels decreased O2 supply to that area.
4. Infarction: Decrease blood supply and lead to insufficient O2.
5. Inflammation: Block blood supply to parts, decreased O2.”

Question 13

What are the Functions of Necrosis?

Answer 13

“Removes damaged cells from an organism

Failure to do so may lead to chronic inflammation.”

Question 14

How is the pH of blood impacted by the distance from the blood vessel?

Answer 14

“pO2 decreases very drastically as the distance from the blood vessel increases.
pH also decreases and becomes more acidic as the pO2 decreases and the pCO2 is relatively higher.”

Question 15

How does necrosis occur?

Answer 15

“1. In the absence of O2 cells cannot generate ATP.

2. This affects the ion pump function.”

Question 16

When is it reversible?

Answer 16

“3. This means the cell cannot control it’s osmolarity.

4. Water moves into the cell causing swelling.
5. Necrosis is a reversible process at this stage.
6. The swelling is reversible.”

Question 17

How would necrosis be reversible at this point?

Answer 17

If we supply O2 to the cell the ion pumps will start working again and the cell will return to normal.

Question 18

When is necrosis no longer reversible?

Answer 18

“1. There is a point of no return, when the influx of water is too much.

2. The nucleus swells.
3. All the organelles expand.
4. The fate of the cell is sealed.
5. Organelles break.
6. Proteins are denatured.
7. The cell membrane breaks down.
8. Haphazard destruction of organelles and nuclear material by enzymes from ruptured lysosomes.
9. Cellular debris stimulates an inflammatory cell response.”

Question 19

Describe the Nuclear Changes in Necrosis?

Answer 19

“1. Chromatin Condensation/shrinkage.

2. Fragmentation of Nucleus.
3. Dissolution of the chromatin by DNase.”

Question 20

What is Basophilia?

Answer 20

“Basophils are a type of white blood cell.
An abnormally high basophil level is called Basophilia.
It can be a sign of chronic inflammation in your body”

Question 21

What Microscopic changes occur to the Cytoplasm during Necrosis?

Answer 21

“1. Opacification: Denaturation of proteins with aggregation.
2. Liquefactive Necrosis: Complete digestion of cells by enzymes causing cell to liquify.”

Question 22

Describe the Biochemical changes that occur due to Necrosis?

Answer 22

“1. Release of enzymes such as Creatine Kinase or Lactate Dehydrogenase.
These enzymes allow us to measure the rate of necrosis

2. Release of proteins such as Myoglobin.

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

Question 23

What is an Astrocytoma?

Answer 23

“Type of cancer of the brain.
They originate in Astrocytes.
This type of tumor doesn’t spread outside the brain and spinal cord and it doesn’t usually affect other organs.
Most common glioma and can occur in most parts of the brain and occasionally in the spinal cord.”

Question 24

Why are Astrocytomas relevant to Necrosis?

Answer 24

Growth of cancer cells cause necrosis.

Question 25

How does necrosis present histologically in the Kidney?

Answer 25

“For the Glomeruli that have undergone necrosis.

• Nuclei have disappeared
• No DNA.
• Maintained shape but nothing inside. “

Question 26

What is Apoptosis?

Answer 26

Selective process for the deletion of superfluous, infected or transformed cells.

Question 27

What are the functions of Apoptosis

Answer 27

“1. Embryogenesis.

2. Metamorphosis.
3. Normal tissue turnover.
4. Endocrine-dependent tissue atrophy.
5. A variety of pathological conditions.”

Question 28

What are the two types of apoptosis?

Answer 28

“Extrinsic:
If signals come from outside the cell

Intrinsic:
If signals come from within the cell”

Question 29

What External Signals can trigger Apoptosis?

Answer 29

“1. Withdrawal of growth factors (e.g. IL-3).

2. Extracellular signals (e.g. TNF).
3. T cell or NK (Natural Killer) (e.g. Granzyme release).”

Question 30

What are Granzymes and what do they do?

Answer 30

“Granzymes are serine proteases that are released by cytoplasmic granules within cytotoxic T cells and natural killer (NK) cells.
They induce programmed cell death in the target cell.
Thus eliminating cells that have become cancerous or are infected with viruses or bacteria.”

Question 31

What Internal Signals can trigger Apoptosis?

Answer 31

“1. DNA damage – p53-dependent pathway.

2. Interruption of the cell cycle.
3. Inhibition of protein synthesis.
4. Viral Infection.
5. Change in redox state.”

Question 32

Give examples of Apoptosis?

Answer 32

“1. Cell death in embryonic hand to form individual fingers.

2. Apoptosis induced by growth factor deprivation:
   eg: Neuronal death from lack of NGF.
3. DNA damage-mediated apoptosis:
   If DNA is damaged due to radiation or chemo therapeutic agents, p53 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 induced by cytotoxic T cells:
   eg: Cellular immune rejection or graft vs. host disease.
6. Death of neutrophils during an acute inflammatory response.
7. Metamorphosis:
   A biological process by which an animal physically develops after birth or hatching.
   Involving a conspicuous and relatively abrupt change in the animal’s body structure through cell growth and
   differentiation.
8. Death of immune cells:
   Both T and B lymphocytes die after depletion of cytokines.
   As well of death of autoreactive T cells in the developing thymus..
9. Normal Tissue Turnover.
10. Endocrine-dependent tissue atrophy:
    Cell death of the milk producing cells when breast feeding is ceased.
11. A variety of pathological conditions.
    Cell death in viral diseases (ie viral hepatitis) as virally infected cells will become factories for infection.”

Question 33

What factors influence the balance of Survival and Apoptosis at the cellular level?

Answer 33

“Survival:

1. Cell - Cell contacts and Cell - Matrix contacts.
2. Growth Factors.
3. Cytokines.

Apoptosis:

1. Distruption of Cell - Cell contacts and Cell - Matrix contacts.
2. Lack of Growth Factors.
3. DNA Damaging Agents.
4. Death Domain Ligands.”

Question 34

What are Caspases?

Answer 34

“Cysteine Aspartate - Specific Proteases:

- Play a central role in the initiation of apoptosis. “

Question 35

How are Proteases activated?

Answer 35

“Most proteases are synthesised as inactive precursors requiring activation.
Usually partial digestion by another protease.”

Question 36

What is the importance of Caspase activation?

Answer 36

“Activation of Caspases ensures that the cellular components are degraded in a controlled manner.
Carrying out cell death with minimal effect on surrounding tissues.”

Question 37

How are Caspases synthesised?

Answer 37

“Caspases are synthesised as inactive zymogens, Pro-Caspases.
That are only activated following an appropriate stimulus.
This post-translational level of control allows rapid and tight regulation of the enzyme.”

Question 38

Describe how Caspases are activated?

Answer 38

“Cleavage:

• Inactive Procaspase Y is cleaved into a Small Subunit, a Large Subunit and a Prodomain.
• This is catalysed by the enzyme Active Caspase X.

Dimerisation:

• The two subunits that were cleaved from Inactive Procaspase Y, the large and small subunits dimerise.
• When these two subunits dimerise they form Active Caspase Y.”

Question 39

Describe the Caspase Cascade?

Answer 39

“1. Apoptosis triggers the activation of the Active Initiator Caspase: Caspase X: 8 or 9.

2. Active Caspase X catalyses the activation of many Effector Caspases: Caspase Y/Caspase Z: 1,3,6,7.
3. Active Caspase X catalyses the activation of Inactive Caspase Y into Active Caspase Y.
   This produces many molecules of Active Caspase Y.
   Active Caspase Y cleave cytosolic proteins containing cysteine or aspartate.
4. Active Caspase Y catalyses the activation of many more Inactive Caspase Z into Active Caspase Z.
   This produces many more molecules of Active Caspase Z.
   Active Caspase Z cleave Nuclear Lamin.
5. As a result of all this cleavage.
   The actin cytoskeleton starts to break down so the cells start to collapse.”

Question 40

What changes from Caspase activation can be seen?

Answer 40

“Characteristic morphological changes:

• Shrinkage.
• Chromatin Condensation.
• DNA Fragmentation.
• Plasma Membrane Blebbing.”

Question 41

What do Blebs consist of?

Answer 41

“All the cell contents are contained in the vesicles/ blebs on the cell surface membrane.
The blebs contain intact organelles such as mitochondria.
They then get coated in sugar and get digested by macrophages.”

Question 42

List the major features of Apoptosis?

Answer 42

“1. Single or few cells selected.

2. Programmed cell death.
3. Irreversible once initiated.
4. Events are energy driven.
5. Cells shrink as the cytoplasm is disassembled.
6. Orderly packing of organelles and nuclear fragments in membrane bound vesicles.
7. New molecules are expressed on the vesicle membranes which stimulate phagocytosis and no inflammatory response.”

Question 43

How does DNA Laddering differ in Apoptotic Cells?

Answer 43

“1. Normal cell DNA is heavy so the DNA gets stuck at the top.

2. There is laddering in apoptotic cells because the nucleosomes are intact.
3. If the cells are dying by necrosis there is no laddering because the fragmentation of necrotic cells is random and don’t have nucleosomes.”

Question 44

Describe the Nuclear Changes in Apoptosis?

Answer 44

“Nuclear chromatin condenses on nuclear membrane.

DNA cleavage.”

Question 45

What changes occur to the Cytoplasm during Apoptosis?

Answer 45

“1. Shrinkage of cell and organelles packed into membrane vesicles.

2. Cell fragmentation as membrane bound vesicles bud off.
3. Phagocytosis of cell fragments by macrophage and adjacent cell.
4. No leakage of cytosolic components.”

Question 46

Describe the Biochemical changes that occur due to Apoptosis?

Answer 46

“Expression of charged sugar molecules on outer surface of cell membranes.
This is recognised by macrophages to enhance phagocytosis.
Protein cleavage by proteases, caspases.”

Question 47

How many substrates are there for Caspases?

Answer 47

“Hundreds of substrates for activated caspases.
Substrates fall into most classes of important genes.

Substrates and their Function:

• Lamin A and B: Nuclear envelope
• PARP: DNA repair
• DNA-PK: DNA repair
• Toposiomerase II: DNA replication
• Raf-1: Signaling
• Akt/PKB: Cell survival
• STAT1: Signaling
• eIF4: Translation “

Question 48

How do we activate the Initiator Caspases?

Answer 48

“By Induced Proximity.

For Example:

1. In response to receptor dimerization upon ligand binding.
2. Cytochrome C release from the mitochondria.”

Question 49

Describe how Ligand Induced Dimerisation occurs?

Answer 49

“This is the Extrinsic Pathway of Apoptosis:
The Receptor has two domains:
1. The Ligand Binding Domain is in the extracellular fluid.
2. The Death Domain forms dimers with the Death Adaptor Protein.

The Death Adaptor Protein has two domains:

1. The Death Domain forms dimers with the Receptor.
2. The Death Effector Domain which can bind to Procaspase - 8. “

Question 50

What is Cytochrome C?

Answer 50

“Mitochondrial Matrix Protein:

• Known for many years to be released in response to oxidative stress by a ‘permeability transition’.
• Any inducers of permeability transition also eventually induce apoptosis.”

Question 51

What is APAF-1 and what domains does it have?

Answer 51

“Apoptotic Protease Activating Factor Protein in the Cytoplasm which has 4 domains:

1. Cytochrome C binding site.
2. APAF Domain.
3. Caspase Recruitment Domain (CARD).
4. Procaspase 9 binds to the CARD domain.”

Question 52

Describe Cytochrome C - induced Apoptosis?

Answer 52

“This is the Intrinsic Pathway of Apoptosis:

1. When Cytochrome C is released from the mitochondria.
2. The Cytochrome C brings together two molecules of APAF-1.
3. The APAF-1 molecules bring together two Procaspase 9 molecules
4. Due to them being close together they can cleave each other (autoproteolysis) and active Caspase 9 is produced. “

Question 53

What proteins regulate the release of Cytochrome C?

Answer 53

“By the bcl-2 family of Genes:

1. Anti - Apoptotic:
   - bcl-2.
   - bcl-XL.
2. Pro - Apoptotic:
   - Bax.
   - Bad.
   - Bid.”

Question 54

What can bcl-2 Proteins form?

Answer 54

“They form dimers.

They can form Homodimers with themselves.”

Question 55

Which protein facilitates the release of Cytochrome C?

Answer 55

“1. Bax is a pro-apoptotic protein that forms dimers with itself.

2. It sits on the mitochondrial membrane and forms a pore in the middle.
3. It allows Cytochrome C to cross the membrane and leave the cell.”

Question 56

Which protein limits the release of Cytochrome C?

Answer 56

“1. In healthy cells bcl-2 makes dimers with bax.

2. It makes a lid on the pore so the Cytochrome C cannot escape.”

Question 57

What removes the bcl-2 block from Bax?

Answer 57

“1. If the cell is not receiving the Survival Signals.

2. Bad is not phosphorylated.
3. So Bad remains activated.
4. Activated Bad competes for Bax binding with bcl-2.
5. This causes the bcl-2 ‘lid’ to be removed and Cytochrome C leaves the cell.
6. Caspase 9 is activated.”

Question 58

When there are survival signals how is the release of Cytochrome C stopped?

Answer 58

“1. PKB/Akt responds to survival signals which makes the Kinase Active.

2. PKB phosphorylates its’ substrate Bad thus inactivating it.
3. So Bad can no longer compete for Bax binding with bcl-2”

Question 59

How can intracellular stress lead to Apoptosis?

Answer 59

“1. Intracellular stress causes transcription by p53.

2. One of the genes it transcribed is bax.
3. The molecules of bax in the cell increase and more pores on the membrane are formed.
4. Bax proteins insert themselves into the membrane.
5. Cytochrome C gets released and cell death is induced.”

Question 60

Describe how mutations in p53 can cause Cancer?

Answer 60

“Mutations in the p53 gene are the most common mutations in cancer:

• Some mutations destroy the ability of p53 to induce apoptosis.
• Cancer treatments are usually done by damaging the DNA and the aim is to trigger p53 to start apoptosis.
• In some cancers p53 is mutated so they are resistant to treatment.”