L4 - Resisting Cell Death Flashcards
Describe some of the growth morphological changes that occur during apoptosis
Blebbing
Detachement
Rapid shape change
Nuclear fragmentation
What compound can induce apoptosis
How does it do this
Staurosporine
Versions mimic ATP which binds to Chk1 in its ATP binding pocket
What are the first molecular characteristics of apoptosis
Chromosomal fragmentation
Nuclear envelope breakdown
Describe how you would see chromosomal fragmentation
Chromosomal DNA (whole)
When fragmented it will run
Can stain with ethidium bromide
Describe the composition of the nuclear lamina
Mesh = lamina made of lamins (protein)
How can we detect nuclear envelope breakdown
Can detect cleaved lamins on a blot
What was used to interrogate the molecular mechanisms for apoptosis
Cell free systems
Describe the formation of the cell free systems
take from a frog - multiple divisions without any cell growth
Crude cytoplasmic fraction can reconstiutude organelles
Take the extract made of eggs and egg naked chromatin
DNA will form chromatin and acquire a nuclear envelope - which is lifelike
Nuclear pores present
What did Newmeyer suggest
Cytochrome C is responsible to initiate the intrinsic apoptotic cascade
This is predominanlty involved in the electron transport chain
What did Lazebnik suggest
Initiator and eecutioner caspases drive apoptosis
What is a caspase
Cystenine and aspartate acid dependent protease
What was the founding member of the caspase family
Interleukin Beta-converting enzyme
What are caspase inhibitors used to treat e
Epilepsy
What was the first experiment used to determine the order of events in apoptosis
Using a kinetic (temporal) analysis of CytC release indicates the order of the molecular events in apoptosis
How does the AnnexinV assay work?
AnnexinV binds PLs not usually at the cell surface - this change occurs during apoptosis
During apoptosis what happens to the peremability of the plasma membrane
PM becomes permeable to small molecules
How can the change in peremabililty of the PM be assayed
Bathe cells in DAPI
Normally cant get into cells
If PM permeable then DAPI will bind to the DNA and fluoresce
What technique was used by Goldstein to label Cyt-C
Label using GFP
How did Goldstein induce apoptosis in the cells
Using a high dose of UV rad
What was the main conclusion from Goldsteins expts
That CytC is release during apoptosis
What was the next step in Goldsteins experiments
What quesiton was this seaking to adress
To inhibit caspases
Ask whether caspases cause the release of cytochrome C
What were Goldsteins conclusions when using a caspase inhibitor
No change in the release of cytochrome C with or without a caspase inhibitor suggests that Cyt C is upstream of the activation of caspases
Cytochrome C release is a
PASSIVE PROCESS
How can the integrity of the mitochondrial membrane be measured
Intact membrane will have an intact membrane potential v
How can we determine if the mt vm is intact
Use a positive ion which is permeable to the outer membrane - if intact then should see movement in
What is TMRE
What is it used for?
Fluoro ester
Aggregates in the mitochondria when the membrane potential is intact
In cells not undergoing apoptosis what can be said about the TMRE staining
Colocalisses with CytC
Indicates an intact membrane potential - which we would expect
In cells undergoign apoptosis what can be said about TMRE and CytC staining
What does this suggeset
No colocalisation
Mt membrane potential (integrity) is lost
Describe the colocalisation experiments in the presence of zFAD
What are the conclusions that can be drawn from this
Mitochondrial membrane potential intact
Cyt C is still being released
Mt membrane potential is independent from the release of cytochrome C
What is the failure of the mitochondrial membrane potential driven by
Caspases
What is Z-FAD
A caspase inhibitor
What gene provides some insight into the release of CytC
B cell lymphoma gene 2 (BCL2)
How is BCL2 created
What is the effect of this
Reciprocal translocation between chromosomes 14 and 18
Gene now under control of a strong promoter - high expression levels
If you express BCL2 in mice do they all get cancer
NO
What is myc involved in
Transcribing genes involved in proliferation
What is the effect of combining Myc and BCL2 in a mouse
What does this suggest
Much much worse
Suggests that Myc has a role in enhancing BCL2
What is seen when lymphocytes are cultured
They have a natural tendency to induce the apoptotic program
Describe what is seen in cultured lymphocytes under the following experimental condition
Control
Slow apoptosis - lymphocytes have a natural tendency to induce apoptosis
Describe what is seen in cultured lymphocytes under the following experimental condition
Myc
Proliferation and apoptosis
Describe what is seen in cultured lymphocytes under the following experimental condition
BCL2
No apoptosis
Describe what is seen in cultured lymphocytes under the following experimental condition
BCL2 and Myc
Proliferation and no apoptosis
THIS IS THE CIRCUMSTANCE WHICH PRODUCES CANCER
Relate the presence of Myc and BCL2 to the sweet spot hypothesis
Increases ‘cancer zone’ upwards
Where does BCL2 localise to
How is this seen
Outer mitochondrial membrane
Seen since it colocalises wth MOM
What different domains does BCL2 have
Transmembrane domain - is inserted into the outer mitochondrial membrane
BCL2 homology domains
What can be said of the different family members of the BCL2 family
There are multiple that have different effects
What are the two groups of BCL2 familiy members
PRO APOPTOTIC
PRO SURVIVAL
What are examples of pro apoptotic family members
Bax family
BH3 only family
What are examples of pro survival family members
BCL2
Describe how proapoptotic and prosruvival genes work
Proapoptotic genes are able to interact ( slot into) pro survival genes
How does Bax (pro apoptotic) leads to and promote apoptosis
Bax into the Mt membrane forms a pore which allows the efflux of cytochrome C
How to pro surival genes block apoptosis
Block the pore formed by pro apoptotic genes leading to CytC remaining in the Mt
BAK is
A proapototic gene
Describe how BAK could be visualised
Using gold coated particles designed to recognise BAK
Describe the localisation of BAK in the absence of any apoptotic stimuli
What about in the presence of apoptotic stimuli
Present on the outer mitochondrial membrane
DIFFUSE
Apply staurosporine
Aggregation occurs
Generally what do apopotitic stimuli cause for the pro apoptotic genes
Aggregation
Give some examples of pro apoptotic stimuli
Cytokine deprivation
Anoikis
p3
What is meant by the sweet spot mechanism in the context of apoptosis
intricate balance of pro apoptotic and pro survival genes which determines the fate of the cell
Give some examples of the proteins that apoptotic signals converge throguh
Puma
Noxa
What is the effect onf Puma and Noxa
Activated in response to apoptotic signals
Inhibition of BCL2 (pro survival)
Which relieves the inhibition on Bax/Bak
Apoptosis
____ in the membrane activates caspases
Bax/Bak
If BCL2 is present then what will occur
Inhibition of Bax/Bak - survival
What is the effect on BIM on BCL2
Bim turned on in response to apoptotic stimuli
Binds to and inhibts BCL2
Relieves the inhibition on Bax
Bax tirggers caspase activation
How does intermediate Ras signalling (to cause proliferation) promote survival
Ras-GTP activates P45 MAP kinase (ERK)
ERK causes phosphorylation and proteolytic destruction of BIM
So now BCL2 inhibits Bax and growth can occur
How does prolonged Ras signalling lead to death
Prolonged Ras causes the activation of P38
Phosphorylation of BCL2 - eliminates function
BAX no longer inhibited
APOPTOSIS
How was the coupling device linking CytC to caspase activation discovered
Extract of apoptotic cells and expose to recombinant caspase
Purify through column chromatography
Multiple factors found - APAF-1 and APAD-3
What is the current molecular model for how APAF-1 works
With caspase - dome on top
Cytochorme C binds to the spokes to swich on pro-caspase 9 - which sits on top of the dome
Caspase 9 recruited to hub and spoke APAF-1 which only assembles in the presence of cytochrome C
Describe how genotoxic stress causes apoptosis
Work through p53 Transcription factor induces Noxa and Puma Inhibit BCL2 No inhibition of Bax Caspase activation
What percentage of tumours have mutations in the p53/noxa axis
90%
