Cell damage and cell death

Question 1

What are the main causes of cell damage/ death?

Answer 1

• Genetic - aneuploidy, indels, increased fragility, failure of repair, inborn errors
• Trauma - interruption of blood supply, direct rupture of cells, foreign agents
• Infection - toxins, competition, IC replication
• Inflammation - trauma, embolism, atherosclerosis, vasculitis
• Physical - irradiation, heat, cold
• Chemical - acids, enzymes, metabolism interference

Question 2

What are the 3 basic mechanisms that cause cell death?

Answer 2

• Necrosis = commonest, occurs after stresses like ischaemia, trauma, chemical injury
• Apoptosis = programmed cell death; eliminate cells that are no longer of use as have already carried out their function
• Autophagic cell death = degradation of normal proteins involved in cellular remodelling during metamorphosis, aging, differentiation and abnormal proteins that would otherwise accumulate following toxin exposure, cancer or disease

Question 3

What are the causes of necrosis?

Answer 3

• Usually lack of blood supply to cells or tissues from:

- Injury, infection, cancer, infarction and inflammation

Question 4

Why do cells need to be close to blood supply?

Answer 4

• oxygen can only diffuse so far through tissues without being metabolised
• Need to be within mms of supply
• The further away, the quicker the PO2 drops off

Question 5

What are the main features of necrosis?

Answer 5

• Whole groups of cells are affected by injurious agent or event
• Reversible swelling occurs from energy deprivation (cant produce enough ATP due to lack of o2). This causes influx of water as no ATP to control ion pumps
• Leads to irreversible swelling
• Haphazard destruction of organelles and nuclear material by enzymes from ruptured lysosomes
• Eventually cell lyses and disintegrates
• Cellular debris causes inflammation

Question 6

What is the microscopic appearance of necrosis?

Answer 6

Nuclear changes

• Chromatin condensation
• Fragmentation of nucleus
• Dissolution of chromatin by DNAse, causing a fading in the basophilia of chromatin

Cytoplasmic changes

• Opacification - denaturation of proteins and aggregation
• Complete digestion of cells by enzymes causing liquefaction

Biochemical changes

• Release of enzymes such as creatine kinase or lactate dehydrogenase
• Release of proteins such as myoglobin

Question 7

Give 4 examples of clinical investigations associated with cell death

Answer 7

• Muscular dystophy - damaged muscles release creatine kinase and lactate dehydrogenase
• heart attack - damaged muscle cells release lactate dehydrogenase
• Bone and liver disease - release ALP and LDH isoforms
• haemolytic anaemia - damaged RBCs release LDH1/2

Question 8

What is an astrocytoma?

Answer 8

Aggressive brain tumours

Question 9

What are the different types of necrosis?

Answer 9

• Coagulative necrosis = seen in hypoxic environments. Cell outline remains after cell death
• Liquefactive necrosis = cellular destruction and pus formation
• Caseous necrosis = mix of coagulative and liquefactive
• Fatty necrosis = lipase action on fatty tissues
• Fibrinoid necrosis = immune-mediated vascular damage causes deposition of fibrin-like material in arterial walls (appear smudgy and acidophilic in LM)

Question 10

What is the function of necrosis?

Answer 10

• Removes damaged cells from an organism

- Failure to do so may lead to chronic inflammation

Question 11

What are the functions of apoptosis?

Answer 11

• Deletion of superfluous, infected or transformed cell - programmed cell death
• Involved in embryogenesis, metamorphosis, normal tissue turnover, endocrine-dependent tissue atrophy and a variety of pathological conditions

Question 12

Give some examples of apoptosis

Answer 12

• Cell death in hand to form fingers
• Apoptosis induced by GF deprivation
• DNA damage-mediated apoptosis (by p53)
• Cell death in tumours causing regression
• Cell death in viral diseases
• Cell death induced by CTLs eg GvHD
• Death of neutrophils during acute inflammation
• Death of lymphocytes after depletion of cytokines

Question 13

What are the 2 types of apoptosis?

Answer 13

• Intrinsic - factors within the cell cause its self-destruction eg DNA damage, cycle interruption, viral infection, inhibition of protein synthesis
• Extrinsic - external factors cause the cell’s self-destruction eg withdrawal of GFs, EC signals (TNF), T-cells/NK

Question 14

What are caspases?

Answer 14

• Cysteine proteases that play a central role in initiating apoptosis - cleave between cysteine and aspartate
• Most proteases are synthesised as inactive precursors requiring activation

Question 15

How are caspases activated?

Answer 15

• Inactive procaspase Y will be activated by the cleavage of the prodomain - done by active caspase X
• The small subunit then moves alongside to make active caspase Y
• Once one caspase becomes activated, it activates other procaspases, which will then activate more molecules
• these will degrade a number of substrates such as cytosolic proteins and actin cytoskeleton (causes cell to collapse) then cleave the nuclear lamins and envelope

Question 16

What is a morphological difference between apoptosis and necrosis?

Answer 16

• In apoptosis, can see that when the cell collapses, bits of mitochondria and nuclear envelope will bud off and will be engulfed by phagocytes nearby
• This is a quite clean process to get rid of the apoptotic cells, whereas in necrosis there is no loss of cell contents

Question 17

What difference will there be between apoptosis and necrosis in electrophoresis?

Answer 17

• Can look at DNA ladder
• In apoptosis, each fragment of DNA on the ladder is the size of DNA wrapped around the nucleosome - DNA is cleaved around the nucleosomes
• In necrosis, the DNA gives a smear. Lots of enzymes released so the DNA can be cleaved wherever, randomly
• Can tell if cell died through N or A

Question 18

What are the main features of apoptosis?

Answer 18

• Single or few cells selected
• programmed cell death
• irreversible once initiated
• Events are energy driven
• cells shrink as cytoskeleton is disassembled
• orderly packaging of organelles and nuclear fragment in vesicles
• new molecules expressed on vesicle membranes stimulate phagocytosis - no inflammation

Question 19

What is the microscopic appearance of apoptosis

Answer 19

Nuclear changes

• nuclear chromatin condenses on nuclear membrane
• DNA cleavage

Cytoplasmic changes

• Shrinkage of cell, organelles packaged
• Cell fragmentation, vesicles bud off
• Phagocytosis of fragments by macrophage
• No leakage of cytosolic components

Biochemical changes

• Expression of charged sugar molecules on outer and inner surface of membranes (recognised by macrophage and enhance phagocytosis)
• Expression of phosphatidylserine on EC leaflet of apoptotic cell
• Protein cleavage by caspases

Question 20

How do we activate the initiator caspases?

Answer 20

• by induced proximity
• Eg in response to receptor dimerisation upon ligand binding
  or Cytochrome C release from mitochondria

Question 21

What happens in ligand-induced dimerisation?

Answer 21

• Receptor on cell membrane - ligand binding domain on outside, death domain inside
• Death domains can form dimers with the death domains on adaptor proteins
• these adaptor proteins have death effector domains, which can form dimers with the death effector domains on procaspase 8

Question 22

How does TNF induce the formation of a death inducing signalling complex (DISC)?

Answer 22

• TNF recruits and brings together many different receptor molecules, which bring death adaptor proteins, which bring together procaspases
• Collection of procaspases close together starts autoproteolysis

Question 23

What is cytochrome C?

Answer 23

• mitochondrial matrix protein
• known for many years to be released in response to oxidative stress by a “permeability transaction”
• any inducers of the permeability transition also eventually induce apoptosis

Question 24

What are the main players in cytochrome c induced apoptosis?

Answer 24

• Cy C can bind APAF, which contains a Cy C binding site and a caspase recruitment domain (CARD)
• This CARD can interact with procaspase 9 and make a formation of dimers
• Forms active caspase enzyme, starting autoproteolysis, triggering death

Question 25

How is the release of cytochrome C regulated?

Answer 25

• Mixture of anti-apoptotic signals (bcl-2, bcl-XL etc) and pro-apoptotic signals (Bax, Bad, Bid etc)

Question 26

What is Bax?

Answer 26

• A pro-apoptotic protein inserted into the mitochondrial membrane
• It can form dimers with itself and makes a pore in the membrane - allowing cytochrome c to move from the inside to outside
• In healthy cells, this is blocked by Bcl-2 (anti-apoptotic), blocks the channel by forming a dimer with Bax

Question 27

What happens in survival factor withdrawal?

Answer 27

• If cells dont have the right GFs, they will commint suicide
• Survival signals check that the cell is ok
• If it is, then survival signals activate Akt/PKB - Bad is phosphorylated and so does nothing
• If the cell stops getting survival signals, Akt/PKB kinases arent active, so cannot phosphorylate Bad.
• Bad removes Bcl-2, allowing cytochrome c to be released
• Cytochrome C brings procaspases together -> death

Question 28

What happens in the scenario of DNA damage?

Answer 28

• DNA damage causes intracellular stress
• p53 becomes active - a TF that binds to DNA promoters such as the Bax promoter
• Makes more Bax, but has same level of bcl-2
• Means that there are new pores in the mitochondria, allowing cytochrome c to be released
• Brings APAFs/ procaspase 9 together -> cell death

Question 29

What happens if there are mutations in p53?

Answer 29

• Mutations in p53 are most common mutations in cancer
• Some mutations destroy the ability of p53 to induce apoptosis
• Chemo - induce DNA damage to allow cells to apoptose
• Tumours with p53 mutations will be resistant to chemo