Cell Damage and cell death Flashcards
What is cell death caused by?
Three basic mechanisms:
- necrosis
- apoptosis
- autophagic cell death
Necrosis
most common cause of cell death
occurs after stresses such as ischaemia, trauma, chemical injury
removed damaged cells from an organism
Apoptosis
Programmed cell death
Designed to eliminate unwanted host cells
affected by a dedicated set of gene products
Autophagic cell death
Autophagy is responsible for:
>degradation of normal proteins involved in cellular remodelling found during metamorphosis, ageing and differentiation
>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
What causes necrosis?
Necrosis is usually caused by a lack of blood supply to cells or tissues, e.g. due to:
- Injury
- Infection
- Cancer
- Infarction
- Inflammation
How does a lack of blood supply cause necrosis of cells?
Due to the lack of blood supply:
- Cells don’t receive oxygen
- ATP not generated- therefore no energy and no oxidative phosphorylation
- Ion pumps at cell membrane stop working in absence of ATP
- Water balance in cells not regulated
- Cells swell because water begins to leak into the cells
- Organelles also swell
- Cell bursts/disintegrates, releasing intracellular components including enzymes from lysosomes which can digest the cell or nearby cells (e.g. proteases, glucosidases, lipases etc.)
- Cellular debris stimulates an inflammatory response
Reversibility of necrosis
Necrosis is reversible, however if swelling is too extreme/massive, it can become irreversible.
Microscopic Appearance of Necrosis
There are nuclear, cytoplasmic and biochemical changes
Nuclear changes in necrosis
1) Chromatin condensation/shrinkage
2) Fragmentation of nucleus
3) Dissolution of the chromatin by DNase
Cytoplasmic changes in necrosis
1) Opacification: denaturation of proteins with aggregation
2) Liquefactive Necrosis: digestion of cells by enzymes causing cell to liquefy
Biochemical changes in necrosis
1) Release of enzymes (e.g. creatine kinase or lactate dehydrogenase)
2) Release of proteins (e.g. myoglobin)
>These biochemical changes are useful in the clinic to measure the extent of tissue damage
In necrotic cells, the nucleosomes are…
denatured due to the release of enzymes from lysosomes
Brain cancer associated with necrotic tissue
astrocytoma
-cancer cells damage nearby tissue which undergo necrosis, becoming more opaque (darker colour)
Difference between normal and necrotic kidney
Necrotic glomeruli in kidneys is apparent during staining where nuclei are not visible (due to the degradation).
Main function of necrosis is to…
remove damaged cells from an organism
-whole groups of cells are affected
Failure to remove damaged cells from an organism by necrosis…
may lead to chronic inflammation
Function of apoptosis
Selective process for the deletion of superfluous (unnecessary), infected or transformed cells
-because it is selective, it does not affect a whole group of cells
What is apoptosis involved in?
- Embryogenesis
- Metamorphosis
- Normal tissue turnover
- Endocrine-dependent tissue atrophy (e.g. milk producing cells not needed after breast-feeding)
- A variety of pathological conditions
Examples of apoptosis
· Cell death in embryonic hand to form individual fingers
· Apoptosis induced by growth factor deprivation (neuronal death from lack of NGF)
· DNA damage-mediated apoptosis. If DNA is damaged due to radiation or chemotherapeutic agents, p53 accumulates. This arrests the cell cycle enabling the cell to repair the damage. If the repair process fails, p53 triggers apoptosis.
· Cell death in tumours causing regression
· Cell death in viral diseases (i.e. viral hepatitis)
· Cell death induced by cytotoxic T cells (i.e. cellular immune rejection or graft vs host disease)
· Death of neutrophils during an acute inflammatory response
· Death of immune cells (both T and B lymphocytes) after depletion of cytokines as well as death of autoreactive T cells in the developing thymus
Factors which promote apoptosis
· Disruption of cell-cell contact and/or cell-matrix contact
· Lack of growth factors
· DNA damaging agents
· Death domain ligands
Factors which promote cell survival
· Cell-cell contact and/or cell-matrix contact
· Growth factors
· Cytokines
Types of apoptosis
intrinsic and extrinsic
Intrinsic apoptosis
Apoptosis triggered by intracellular signals:
- DNA damage- p53 dependent pathway
- Interruption of the cell cycle
- Inhibition of protein synthesis
- Viral Infection
- Change in redox state
Extrinsic apoptosis
Apoptosis triggered by extracellular signals:
- Withdrawal of growth factors (e.g. IL-3)
- Extracellular signals (e.g. TNF)
- T cell or NK (Natural Killer) (e.g. Granzyme)
What initiates apoptosis?
family of proteases called caspases
What are caspases?
Cysteine Aspartate-specific Proteases which cleave proteins with cysteine and aspartate residues
In what form are most proteases synthesised?
as inactive precursors requiring activation
-usually partial digestion by another protease
Apoptosis is mediated by… (describe the process)
intracellular proteolytic cascade
1) Inactive procaspase Y is activated by active caspase X (initiator caspase 8/9) by cleavage at N-terminal and C-terminal cleavage sites
2) As a result of the cleavage, two parts of the procaspase Y will dimerize, forming the active caspase Y.
3) Active caspase Y (effector caspase) can then go on to degrade further substrates
Caspase cascade
1) Active initiator caspase (i.e. caspase 8 or 9) will activate further procaspases into active caspases.
> these active caspases then go on to cleave cytosolic proteins containing Cysteine and Aspartate residues. As a result of this, the actin cytoskeleton breaks down and therefore the cells break down.
2) Many of these active caspases also activate further caspases (i.e. effector caspases 1,3,6,7) which go on to cleave nuclear lamin protein (essential for the nuclear envelope)
> therefore nuclear envelope is broken down
Apoptosis: Morphological changes
Caspase activation leads to cell shrinkage, chromatin condensation, DNA fragmentation and plasma membrane blebbing.
Actin cytoskeleton is lost during apoptosis, cells become more rounded. Eventually, cell detach from their surface and float in the medium
Why is there no inflammatory response in apoptosis?
Cells start forming blebs (vesicle membranes) which eventually bud from the cell and express new molecules which are recognised by phagocytes/macrophages that engulf them and digest them.
In apoptotic cells, the nucleosomes…
remain intact, but the DNA in between nucleosomes is cleaved
Nucleosomes in necrotic cells vs apoptotic cells
In apoptotic cells, the nucleosomes remain intact, but the DNA in between nucleosomes is cleaved.
In necrotic cells, the nucleosomes are denatured due to the release of enzymes from lysosomes.
Reversibility of apoptosis
Irreversible once initiated
Microscopic Appearance of Apoptosis
There are nuclear, cytoplasmic and biochemical changes
Nuclear changes in apoptosis
1) Chromatin condensation
2) DNA cleavage
Cytoplasmic changes in apoptosis
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 in apoptosis
1) Expression of charged sugar molecules on outer surface of cell membranes (Recognised by macrophages to enhance phagocytosis)
2) Protein cleavage by proteases (caspases)
How is the first initiator caspase activated?
INDUCED PROXIMITY
-in response to receptor dimerisation upon ligand binding (ligand-induced dimerisation)
>extrinsic pathway
-in response to cytochome C release from mitochondria
>intrinsic pathway
Key molecules in extrinsic apoptotic pathway
Transmembrane receptor with two domains, an extracellular ligand binding domain & an intracellular death domain.
Death adaptor protein with two domains, a death domain and a death effector domain. The death domain can form dimers with the death domain of the transmembrane receptor.
Procaspase-8 with two domains, a protease domain and a death effector domain. The death effector domain can interact with the death effector domain of the death adaptor protein.
How is the initiator caspase in the extrinsic pathway activated?
Tumour necrosis factor-TNF (ligand) induces the formation of a death-inducing signalling complex (DISC).
After the interactions between the domains and their close proximity due to the binding of TNF, the procaspase-8 proteins undergo autoproteolysis and as a result, the procaspase-8 proteins become active caspase-8 proteins (initiator caspase).
> This therefore activates the caspase cascade
What is cytochrome C?
a mitochondrial matrix protein released in response to oxidative stress by a “permeability transition”
Key molecules in the intrinsic apoptotic pathway
Cytochrome C- molecule found exclusively within the mitochondria
APAF-1 protein with three domains
- cytochrome C binding site
- APAF domain
-Caspase recruitment domain (CARD)
Procaspase-9 has CARD domain which binds APAF-1 protein
How is the initiator caspase in the intrinsic pathway activated?
Cytochrome C induces the formation of a death-inducing complex.
> a few cytochrome C molecules will bring together APAF-1 molecules
> which in turn bring together procaspase-9 molecules
> close proximity of procaspase-9 molecules induced autoproteolysis, activating procaspase-9 into caspase-9 (initiator caspase)
> caspase cascade then activated
Where is cytochrome C found?
inner mitochondrial membrane
What regulates the release of cytochrome C from the mitochondria?
Bcl-2 family of proteins regulate the release of cytochrome C from the mitochondria.
>Anti-apoptotic: bcl-2, bcl-XL, others
>Pro-apoptotic: Bax, Bad, Bid, others
Expression of anti-apoptotic Bcl-2 proteins…
cells will survive
Expression of pro-apoptotic Bcl-2 proteins…
cells will die
Where is Bax located?
outer mitochondrial membrane
Function of Bax
Pro-apoptotic:
1) Bax forms homodimers on the mitochondrial membrane
2) Pore is formed which allows cytochrome C to be released from the mitochondria into the cytosol
3) Cytochrome C activates APAF and caspase-9 to initiate apoptosis
Function of Bcl-2
Anti-apoptotic protein which prevents normal healthy cells from undergoing apoptosis:
1) bcl-2 forms heterodimers with Bax and blocks the pore
2) cytochrome C isn’t released from mitochondria
What alters the balance between pro-apoptotic and anti-apoptotic proteins?
- survival signals from the environment
- intracellular stress (e.g. DNA damage)
Effect of survival signals on apoptosis
If the cells are receiving the right survival signals:
- Protein Kinase B/AKT is activated and phosphorylates Bad (pro-apoptotic) to terminate its actions.
If the cells don’t receive appropriate survival signals (survival factor withdrawal):
- Protein Kinase B (AKT) is not activated
- Bad therefore is in the de-phosphorylated form
- Bad competes with Bcl-2 for the binding to Bax complex, and as a result Bad forms a heterodimer with Bcl-2 (anti-apoptotic)
- The block on the Bax complex pore is therefore removed, permitting the release of cytochrome C into the cytosol to initiate apoptosis.
Effect of intracellular stress (DNA damage) on apoptosis
During intracellular stress (e.g. DNA damage):
- p53 (transcription factor) is activated
- p53 initially activates transcription of cell cycle inhibitor p21 to try and stop cell cycle to repair DNA
- if DNA repair does not occur, p53 will activate expression of pro-apoptotic proteins (e.g. Bax)
- increase in Bax gene expression increases Bax levels within the cell, creating more Bax complexes which will insert on the mitochondrial membrane
- More pores are created permitting the release of cytochrome C into the cytosol to initiate apoptosis
p53 and apoptosis
Mutations in the p53 gene are the most common mutations in cancer. Some mutations destroy the ability of p53 to induce apoptosis, leading to cancer.
