Cell Damage and Cell Death Flashcards
Outline the genetic causes of cell damage / death
Genetic
- Abnormal number chromosomes (aneuploidy)
- Abnormal chromosomes (deletions/translocations)
- Increased fragility (Fanconi’s anaemia)
- Failure of repair (Xeroderma pigmentosa)
- Inborn errors (Storage disorders ie. Tay Sachs disease)
Give examples of traumatic damage that can cause cell death and damage
Traumatic Damage
- Interruption of blood supply
- Direct rupture of cells
- Entry of foreign agents
What physical factors can cause cell death and damage?
Physical
- Irradiation
- Heat
- Cold
- Barotrauma
Which chemicals cause cell damage?
Chemical
- Acids/corrosives
- Specific actions e.g. enzymes
- Interference with metabolism e.g. alcohol
How can inflammation lead to cell death and damage?
Inflammation
- Trauma
- Thrombo-embolism
- Atherosclerosis
- Vasculitis
Outline how infection can induce cell death and damage
Infection - Toxic agents - Competition for nutrients - Intracellular replication > viruses/mycobacteria provoking an immune response
What mechanism is responsible for cell death?
Cell death is caused by three basic mechanisms:
- Necrosis
- Apoptosis
- Autophagic cell death
What is necrosis?
Necrosis: most common cause of cell death
Occurs after stresses such as ischemia, trauma, chemical injury
What is apoptosis?
programmed cell death
Designed to eliminate unwanted host cells through activation of a co-ordinated, internally programmed series of events affected by a dedicated set of gene products
What is autophagic cell death?
Autophagy is responsible for degradation of normal proteins involved in cellular remodeling found during metamorphosis, aging and differentiation
Also used for digestion and removal of abnormal proteins that can accumulate following toxin exposure, cancer, or disease
Give an example of autophagy
An example is the death of breast cancer cells induced by Tamoxifen.
What are the causes of necrosis?
Usually caused by lack of blood supply to cells or tissues, e.g.
- injury
- infection
- cancer
- infarction
- inflammation
Explain how lack of blood flow can induce necrosis
pH and po2 levels decrease the further from a vessel - dagerous for cells
Which cells are affected by necrosis?
Whole groups of cells are affected following an injurious agent or event
Outline the process of necrosis
Reversible events proceed irreversible.
- Energy deprivation causes changes. (e.g. cells unable to produce ATP because of oxygen deprivation)
- Cells swell due to influx of water (ATP is required for ion pumps to work).
- Haphazard destruction of organelles and nuclear material by enzymes from ruptured lysosomes.
- Cellular debris stimulates an inflammatory cell response
What are the types of microscopic changes in cell appearance associated with necrosis?
- nuclear changes
- cytoplasmic changes
- biochemical changes
Describe the nuclear changes that occur in cells during necrosis
- Chromatin condensation/shrinkage.
- Fragmentation of nucleus.
- Dissolution of the chromatin by DNAse
What changes occur to cell cytoplasm during necrosis?
- Opacification: denaturation of proteins with aggregation.
- Complete digestion of cells by enzymes causing cell to
liquify (liquefactive necrosis).
What are the biochemical changes seen in necrotic cells?
- Release of enzymes such as creatine kinase or lactate dehydrogenase
- Release of proteins such as myoglobin
What is the significance of the biochemical changes that occur during necrosis?
These biochemical changes are useful in the clinic to measure the extent of tissue damage
What is astrocytoma?
Frequent brain tumour: highly progressive and diagnosis is low
How do astrocytomic cells appear?
Can see the opacification of necrotic tissue due to denaturation of proteins causing a darker appearance
The tumour is evading the normal histology of the brain cells
Describe the differences seen in stained glomeruli of normal and necrotic kidneys
Normal glomeruli; can see dark staining where cell nuclei are present
Necrotic glomerul; doesn’t stain as well as DNA is degraded
What are the functions of necrosis?
Removes damaged cells from an organism
Failure to do so may lead to chronic inflammation
What are the functions of apoptosis?
Selective process for the deletion of superfluous, infected or transformed cells
What other processes is apoptosis heavily involved in?
- Embryogenesis
- Metamorphosis
- Normal tissue turnover
- Endocrine-dependent tissue atrophy
- A variety of pathological conditions
What is the purpose of apoptosis in embryonic hands?
Cell death in the embryonic hand forms individual fingers.
What is the role of growth factor in apoptosis?
Apoptosis can be induced by growth factor deprivation e.g.(neuronal death from lack of NGF)
Explain 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 the repair process fails, p53 triggers apoptosis
Give examples of pathologies that induce apoptosis?
Cell death in tumours causing regression.
Cell death in viral diseases (ie viral hepatitis).
How does the immune system contribute to apoptosis?
Cell death induced by cytotoxic T cells (ie. 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 of death of autoreactive T cells in the developing thymus..
Which factors promote cell survival ?
- cell-cell and/or cell-matrix contacts
- growth factors
- cytokines
Which factors promote apoptosis ?
- disruption of cell-cell and/or cell-matrix contacts
- lack of growth factors
- death domain ligands
- DNA damaging agents
What are the 2 types of apoptosis?
- Intrinsic; triggered from within a cell
- Extrinsic; stems from outside the cell
Outline the intrinsic causes of apoptosis
- DNA damage (p53 dependent pathway)
- Cell cycle interruption
- Protein synthesis inhibition
- viral infection
- change in redox state
Outline the extrinsic causes of apoptosis
- Withdrawal of growth factors (e.g. IL-3)
- Extracellular signals (e,g, TNF)
- T-cell / NKs (e.g. Granzyme)
What are caspases?
(Cysteine Aspartate-specific Proteases)
Caspases are Cysteine Proteases that play a central role in the initiation of apoptosis
How are caspases synthesised?
Most proteases are synthesised as inactive precursors requiring activation (usually partial digestion by another protease).
How is apoptosis regulated?
Apoptosis is mediated by an intracellular proteolytic cascade
Outline how inactive Caspase precursors are activated
- Inactive caspase Y is activated via cleavage at the
amino and carboxyl terminals by active caspase X - Due to the cleavage there are 2 parts of now active
caspase Y which form a dimer - The dimer can go on to degrade further substrates
Describe the caspase cascade
Inside cells active initiator caspase (8/9) leads to procaspase Y activation which goes on to cleave further effector caspases (1,3,6,7)
What are the caspase substrates involved in the apoptotic caspase cascade?
The caspase substrates are as following:
Procaspase Y start to cleave cytosolic proteins (actin filaments, microfilaments)
Effector caspases cleave nuclear lamin
What is the result of caspase (8/9) activation?
Caspase activation leads to characteristic morphological changes of the cell such as shrinkage, chromatin condensation, DNA fragmentation and plasma membrane blebbing.
Describe the morphological changes seen in cells undergoing apoptosis
Seen via TEM (transmission electron micrograph)
- Virtually lost all actin filaments and cells become
rounded blobs
Seen via SEM (scanning electron micrograph)
- Bulges form on cell surface containing intact organelles
(mitochondria etc.)
- Bulges bud off the cells and are recognised by
phagocytes and macrophages - will be engulfed and
removed
What is the major difference in necrosis and apoptosis mechanisms?
Unlike necrosis there is no inflammatory response as budding off cells removed by macrophages
How can DNA fragmentation via gel electrophoresis be used to see apoptotic and necrotic cells?
A population of healthy cells is obtained, and the genomic DNA is purified and treated with an apoptotic agent → DNA ladders form
Describe what we see in DNA fragmentation of apoptotic cells?
During apoptosis we can see an orderly degradation of protein and compounds
How do necrotic cells appear in DNA fragmentation?
In necrosis we see a DNA smear as opposed to a ladder - due to the fact in necrosis all the enzymes are released and degradation occurs at once; no nucleosome particles
Which cells undergo apoptosis?
Single or few cells selected for programmed cell death.
Events are energy driven
Irreversible once initiated
Outline the process of apoptosis
- Cells shrink as the cytoskeleton is disassembled.
- Orderly packaging of organelles and nuclear fragments
in membrane bound vesicles. - New molecules expressed on vesicle membranes
stimulate phagocytosis, no inflammatory response.
How many substrates activate initiator caspase complexes to induce apoptosis?
Apoptosis = “Death by a thousand cuts”
- Hundreds of substrates for activated caspases
- Substrates fall into most classes of important genes
What activates caspases to induce apoptosis?
Initiator caspases are activated by induced proximity
How are caspases brought in close proximity of one another to induce apoptosis?
Extrinsic
- In response to receptor dimerisation upon ligand binding
or
Intrinsic
- Cytochrome C release from the mitochondria.
Outline the substrates involved in the extrinsic ligand-induced dimerisation of caspases
Transmembrane receptor has 2 domains:
- Ligand binding domain on extracellular surface
- Death domain located intracellularly
The Death adaptor protein also has 2 domains:
- death domain
- death effector domain
Cells express all of these components but the system is not functioning until they receive TNF (Tumour necrosis factor)
Outline the mechanism of action of ligand-induced dimerisation
- TNF released
- The transmembrane receptor death domain forms a
dimer with a death adaptor protein - The death effector domain binds to the protease
domain of the initiator caspase (e.g. procaspase 8) - Autoproteolysis of procapsase into caspase 8
What is the role of TNF in the extrinsic pathway of caspase activation?
TNF induces the formation of a death-inducing signalling complex = DISC
TNF allows dimerisation of the ligands to bring adaptor proteins and caspases together.
What is the result of cells being exposed to TNF?
As a result of the caspases being brought in close proximity, autoproteolysis occurs forming active caspase 8 → induces caspase cascade
What is cytochrome c?
Mitochondrial matrix protein that also induces apoptosis
How is cytochrome C released?
Known for many years to be released in response to oxidative stress by a “permeability transition”
Any inducers of the permeability transition also eventually induce apoptosis
What are the substrates for the intrinsic apoptosis pathway?
Cytochrome C from mt. matrix
APAF protein in cell cytoplasm has 3 domains:
- Cytochrome C binding site
- APAF domain
- Caspase recruitment domain (CARD)
Outline the intrinsic apoptosis pathway
- CARD binds procaspase 9 molecules
- Cytochrome c released from mitochondria induces the
formation of a death-inducing complex - Causes dimerization of 2 APAF molecules allowing
procaspase 9 molecules to be in very close proximity - Procaspase 9 molecules in close proximity induce
autoproteolysis and cleave each other to produce active
procaspase 9
What is Procaspase 9?
Procaspase 9 is a precursor expressed in cells; not yet active
How is cytochrome C release regulated?
Cytochrome C release form mitochondria is regulated by a family of bcl-2 proteins
What is bcl-2?
bcl-2 is a member of a 7 multi-gene family in mammals
The bcl-2 family members form dimers
Homodimers with themselves or heterodimers with other family members
What is the role of bcl-2 proteins?
Mainly form dimers with anti-apoptotic proteins favouring life in the cells
When pro-apoptotic dimers form cells undergo apoptosis
Or can have a balance between anti-apoptotic and pro-apoptotic
How do bcl-2 family proteins mediate cytochrome c release?
Within cell cytoplasm (cytochrome C in mitochondrial lumen)
- Bax protein forms homodimers on mitochondrial
membrane with pores in the middle - Pore is large enough to allow cytochrome C
transportation from lumen to cytoplasm to activate
APAF/Caspase 9 inducing apoptosis - Bcl-2 (anti-apoptotic protein) forms a dimer with pro-
apoptotic Bax protein => forms a lid on the pore - Cytochrome C isn’t able to pass through so is
maintained in mitochondria
What is cell survival dependent upon?
Cell survival is dependent on cells receiving the correct survival signals in the right environment
What is the result of correct cell signalling?
If correct signalling is occurring, an intracellular survival signal Akt/PKB is activated to phosphorylate the Bad protein rendering it inactive => cell survives
What is the consequence of incorrect cell signalling?
If survival signals aren’t received, PKB no longer works, Bad is dephosphorylated and is able to dimerise with other family members => apoptosis
How is cell signalling induced apoptosis regulated?
There are equal numbers of pro-apoptotic and antiapoptotic proteins = there is competition between them
Explain how dimeristaion of certain bcl-2 family proteins due to cell signalling can induce apoptosis
Bad can bind to bcl-2; removes lid allowing cytochrome C to pass and activate caspase-9
⇒ apoptosis
What is the significance of P53?
Mutations in the p53 gene are the most common mutations in cancer.
Some mutations destroy the ability of p53 to induce apoptosis.
