L4 - Resisting Cell Death

Question 1

Describe some of the growth morphological changes that occur during apoptosis

Answer 1

Blebbing
Detachement
Rapid shape change
Nuclear fragmentation

Question 2

What compound can induce apoptosis

How does it do this

Answer 2

Staurosporine

Versions mimic ATP which binds to Chk1 in its ATP binding pocket

Question 3

What are the first molecular characteristics of apoptosis

Answer 3

Chromosomal fragmentation

Nuclear envelope breakdown

Question 4

Describe how you would see chromosomal fragmentation

Answer 4

Chromosomal DNA (whole)
When fragmented it will run
Can stain with ethidium bromide

Question 5

Describe the composition of the nuclear lamina

Answer 5

Mesh = lamina made of lamins (protein)

Question 6

How can we detect nuclear envelope breakdown

Answer 6

Can detect cleaved lamins on a blot

Question 7

What was used to interrogate the molecular mechanisms for apoptosis

Answer 7

Cell free systems

Question 8

Describe the formation of the cell free systems

Answer 8

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

Question 9

What did Newmeyer suggest

Answer 9

Cytochrome C is responsible to initiate the intrinsic apoptotic cascade
This is predominanlty involved in the electron transport chain

Question 10

What did Lazebnik suggest

Answer 10

Initiator and eecutioner caspases drive apoptosis

Question 11

What is a caspase

Answer 11

Cystenine and aspartate acid dependent protease

Question 12

What was the founding member of the caspase family

Answer 12

Interleukin Beta-converting enzyme

Question 13

What are caspase inhibitors used to treat e

Answer 13

Epilepsy

Question 14

What was the first experiment used to determine the order of events in apoptosis

Answer 14

Using a kinetic (temporal) analysis of CytC release indicates the order of the molecular events in apoptosis

Question 15

How does the AnnexinV assay work?

Answer 15

AnnexinV binds PLs not usually at the cell surface - this change occurs during apoptosis

Question 16

During apoptosis what happens to the peremability of the plasma membrane

Answer 16

PM becomes permeable to small molecules

Question 17

How can the change in peremabililty of the PM be assayed

Answer 17

Bathe cells in DAPI
Normally cant get into cells
If PM permeable then DAPI will bind to the DNA and fluoresce

Question 18

What technique was used by Goldstein to label Cyt-C

Answer 18

Label using GFP

Question 19

How did Goldstein induce apoptosis in the cells

Answer 19

Using a high dose of UV rad

Question 20

What was the main conclusion from Goldsteins expts

Answer 20

That CytC is release during apoptosis

Question 21

What was the next step in Goldsteins experiments

What quesiton was this seaking to adress

Answer 21

To inhibit caspases

Ask whether caspases cause the release of cytochrome C

Question 22

What were Goldsteins conclusions when using a caspase inhibitor

Answer 22

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

Question 23

Cytochrome C release is a

Answer 23

PASSIVE PROCESS

Question 24

How can the integrity of the mitochondrial membrane be measured

Answer 24

Intact membrane will have an intact membrane potential v

Question 25

How can we determine if the mt vm is intact

Answer 25

Use a positive ion which is permeable to the outer membrane - if intact then should see movement in

Question 26

What is TMRE What is it used for?

Answer 26

Fluoro ester Aggregates in the mitochondria when the membrane potential is intact

Question 27

In cells not undergoing apoptosis what can be said about the TMRE staining

Answer 27

Colocalisses with CytC | Indicates an intact membrane potential - which we would expect

Question 28

In cells undergoign apoptosis what can be said about TMRE and CytC staining What does this suggeset

Answer 28

No colocalisation | Mt membrane potential (integrity) is lost

Question 29

Describe the colocalisation experiments in the presence of zFAD What are the conclusions that can be drawn from this

Answer 29

Mitochondrial membrane potential intact Cyt C is still being released Mt membrane potential is independent from the release of cytochrome C

Question 30

What is the failure of the mitochondrial membrane potential driven by

Answer 30

Caspases

Question 31

What is Z-FAD

Answer 31

A caspase inhibitor

Question 32

What gene provides some insight into the release of CytC

Answer 32

B cell lymphoma gene 2 (BCL2)

Question 33

How is BCL2 created What is the effect of this

Answer 33

Reciprocal translocation between chromosomes 14 and 18 | Gene now under control of a strong promoter - high expression levels

Question 34

If you express BCL2 in mice do they all get cancer

Answer 34

NO

Question 35

What is myc involved in

Answer 35

Transcribing genes involved in proliferation

Question 36

What is the effect of combining Myc and BCL2 in a mouse What does this suggest

Answer 36

Much much worse Suggests that Myc has a role in enhancing BCL2

Question 37

What is seen when lymphocytes are cultured

Answer 37

They have a natural tendency to induce the apoptotic program

Question 38

Describe what is seen in cultured lymphocytes under the following experimental condition Control

Answer 38

Slow apoptosis - lymphocytes have a natural tendency to induce apoptosis

Question 39

Describe what is seen in cultured lymphocytes under the following experimental condition Myc

Answer 39

Proliferation and apoptosis

Question 40

Describe what is seen in cultured lymphocytes under the following experimental condition BCL2

Answer 40

No apoptosis

Question 41

Describe what is seen in cultured lymphocytes under the following experimental condition BCL2 and Myc

Answer 41

Proliferation and no apoptosis THIS IS THE CIRCUMSTANCE WHICH PRODUCES CANCER

Question 42

Relate the presence of Myc and BCL2 to the sweet spot hypothesis

Answer 42

Increases 'cancer zone' upwards

Question 43

Where does BCL2 localise to How is this seen

Answer 43

Outer mitochondrial membrane Seen since it colocalises wth MOM

Question 44

What different domains does BCL2 have

Answer 44

Transmembrane domain - is inserted into the outer mitochondrial membrane BCL2 homology domains

Question 45

What can be said of the different family members of the BCL2 family

Answer 45

There are multiple that have different effects

Question 46

What are the two groups of BCL2 familiy members

Answer 46

PRO APOPTOTIC PRO SURVIVAL

Question 47

What are examples of pro apoptotic family members

Answer 47

Bax family | BH3 only family

Question 48

What are examples of pro survival family members

Answer 48

BCL2

Question 49

Describe how proapoptotic and prosruvival genes work

Answer 49

Proapoptotic genes are able to interact ( slot into) pro survival genes

Question 50

How does Bax (pro apoptotic) leads to and promote apoptosis

Answer 50

Bax into the Mt membrane forms a pore which allows the efflux of cytochrome C

Question 51

How to pro surival genes block apoptosis

Answer 51

Block the pore formed by pro apoptotic genes leading to CytC remaining in the Mt

Question 52

BAK is

Answer 52

A proapototic gene

Question 53

Describe how BAK could be visualised

Answer 53

Using gold coated particles designed to recognise BAK

Question 54

Describe the localisation of BAK in the absence of any apoptotic stimuli What about in the presence of apoptotic stimuli

Answer 54

Present on the outer mitochondrial membrane DIFFUSE Apply staurosporine Aggregation occurs

Question 55

Generally what do apopotitic stimuli cause for the pro apoptotic genes

Answer 55

Aggregation

Question 56

Give some examples of pro apoptotic stimuli

Answer 56

Cytokine deprivation Anoikis p3

Question 57

What is meant by the sweet spot mechanism in the context of apoptosis

Answer 57

intricate balance of pro apoptotic and pro survival genes which determines the fate of the cell

Question 58

Give some examples of the proteins that apoptotic signals converge throguh

Answer 58

Puma | Noxa

Question 59

What is the effect onf Puma and Noxa

Answer 59

Activated in response to apoptotic signals Inhibition of BCL2 (pro survival) Which relieves the inhibition on Bax/Bak Apoptosis

Question 60

____ in the membrane activates caspases

Answer 60

Bax/Bak

Question 61

If BCL2 is present then what will occur

Answer 61

Inhibition of Bax/Bak - survival

Question 62

What is the effect on BIM on BCL2

Answer 62

Bim turned on in response to apoptotic stimuli Binds to and inhibts BCL2 Relieves the inhibition on Bax Bax tirggers caspase activation

Question 63

How does intermediate Ras signalling (to cause proliferation) promote survival

Answer 63

Ras-GTP activates P45 MAP kinase (ERK) ERK causes phosphorylation and proteolytic destruction of BIM So now BCL2 inhibits Bax and growth can occur

Question 64

How does prolonged Ras signalling lead to death

Answer 64

Prolonged Ras causes the activation of P38 Phosphorylation of BCL2 - eliminates function BAX no longer inhibited APOPTOSIS

Question 65

How was the coupling device linking CytC to caspase activation discovered

Answer 65

Extract of apoptotic cells and expose to recombinant caspase Purify through column chromatography Multiple factors found - APAF-1 and APAD-3

Question 66

What is the current molecular model for how APAF-1 works

Answer 66

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

Question 67

Describe how genotoxic stress causes apoptosis

Answer 67

``` Work through p53 Transcription factor induces Noxa and Puma Inhibit BCL2 No inhibition of Bax Caspase activation ```

Question 68

What percentage of tumours have mutations in the p53/noxa axis

Answer 68

90%