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BMS379 - Cancer Biology > L4 - Resisting Cell Death > Flashcards

L4 - Resisting Cell Death Flashcards

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1
Q

Describe some of the growth morphological changes that occur during apoptosis

A

Blebbing
Detachement
Rapid shape change
Nuclear fragmentation

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2
Q

What compound can induce apoptosis

How does it do this

A

Staurosporine

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

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3
Q

What are the first molecular characteristics of apoptosis

A

Chromosomal fragmentation

Nuclear envelope breakdown

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4
Q

Describe how you would see chromosomal fragmentation

A

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

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5
Q

Describe the composition of the nuclear lamina

A

Mesh = lamina made of lamins (protein)

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6
Q

How can we detect nuclear envelope breakdown

A

Can detect cleaved lamins on a blot

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7
Q

What was used to interrogate the molecular mechanisms for apoptosis

A

Cell free systems

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8
Q

Describe the formation of the cell free systems

A

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

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9
Q

What did Newmeyer suggest

A

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

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10
Q

What did Lazebnik suggest

A

Initiator and eecutioner caspases drive apoptosis

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11
Q

What is a caspase

A

Cystenine and aspartate acid dependent protease

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12
Q

What was the founding member of the caspase family

A

Interleukin Beta-converting enzyme

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13
Q

What are caspase inhibitors used to treat e

A

Epilepsy

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14
Q

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

A

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

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15
Q

How does the AnnexinV assay work?

A

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

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16
Q

During apoptosis what happens to the peremability of the plasma membrane

A

PM becomes permeable to small molecules

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17
Q

How can the change in peremabililty of the PM be assayed

A

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

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18
Q

What technique was used by Goldstein to label Cyt-C

A

Label using GFP

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19
Q

How did Goldstein induce apoptosis in the cells

A

Using a high dose of UV rad

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20
Q

What was the main conclusion from Goldsteins expts

A

That CytC is release during apoptosis

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21
Q

What was the next step in Goldsteins experiments

What quesiton was this seaking to adress

A

To inhibit caspases

Ask whether caspases cause the release of cytochrome C

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22
Q

What were Goldsteins conclusions when using a caspase inhibitor

A

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

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23
Q

Cytochrome C release is a

A

PASSIVE PROCESS

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24
Q

How can the integrity of the mitochondrial membrane be measured

A

Intact membrane will have an intact membrane potential v

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25
Q

How can we determine if the mt vm is intact

A

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

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26
Q

What is TMRE

What is it used for?

A

Fluoro ester

Aggregates in the mitochondria when the membrane potential is intact

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27
Q

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

A

Colocalisses with CytC

Indicates an intact membrane potential - which we would expect

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28
Q

In cells undergoign apoptosis what can be said about TMRE and CytC staining

What does this suggeset

A

No colocalisation

Mt membrane potential (integrity) is lost

29
Q

Describe the colocalisation experiments in the presence of zFAD

What are the conclusions that can be drawn from this

A

Mitochondrial membrane potential intact
Cyt C is still being released

Mt membrane potential is independent from the release of cytochrome C

30
Q

What is the failure of the mitochondrial membrane potential driven by

A

Caspases

31
Q

What is Z-FAD

A

A caspase inhibitor

32
Q

What gene provides some insight into the release of CytC

A

B cell lymphoma gene 2 (BCL2)

33
Q

How is BCL2 created

What is the effect of this

A

Reciprocal translocation between chromosomes 14 and 18

Gene now under control of a strong promoter - high expression levels

34
Q

If you express BCL2 in mice do they all get cancer

A

NO

35
Q

What is myc involved in

A

Transcribing genes involved in proliferation

36
Q

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

What does this suggest

A

Much much worse

Suggests that Myc has a role in enhancing BCL2

37
Q

What is seen when lymphocytes are cultured

A

They have a natural tendency to induce the apoptotic program

38
Q

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

Control

A

Slow apoptosis - lymphocytes have a natural tendency to induce apoptosis

39
Q

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

Myc

A

Proliferation and apoptosis

40
Q

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

BCL2

A

No apoptosis

41
Q

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

BCL2 and Myc

A

Proliferation and no apoptosis

THIS IS THE CIRCUMSTANCE WHICH PRODUCES CANCER

42
Q

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

A

Increases ‘cancer zone’ upwards

43
Q

Where does BCL2 localise to

How is this seen

A

Outer mitochondrial membrane

Seen since it colocalises wth MOM

44
Q

What different domains does BCL2 have

A

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

45
Q

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

A

There are multiple that have different effects

46
Q

What are the two groups of BCL2 familiy members

A

PRO APOPTOTIC

PRO SURVIVAL

47
Q

What are examples of pro apoptotic family members

A

Bax family

BH3 only family

48
Q

What are examples of pro survival family members

A

BCL2

49
Q

Describe how proapoptotic and prosruvival genes work

A

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

50
Q

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

A

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

51
Q

How to pro surival genes block apoptosis

A

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

52
Q

BAK is

A

A proapototic gene

53
Q

Describe how BAK could be visualised

A

Using gold coated particles designed to recognise BAK

54
Q

Describe the localisation of BAK in the absence of any apoptotic stimuli

What about in the presence of apoptotic stimuli

A

Present on the outer mitochondrial membrane
DIFFUSE

Apply staurosporine

Aggregation occurs

55
Q

Generally what do apopotitic stimuli cause for the pro apoptotic genes

A

Aggregation

56
Q

Give some examples of pro apoptotic stimuli

A

Cytokine deprivation
Anoikis
p3

57
Q

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

A

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

58
Q

Give some examples of the proteins that apoptotic signals converge throguh

A

Puma

Noxa

59
Q

What is the effect onf Puma and Noxa

A

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

60
Q

____ in the membrane activates caspases

A

Bax/Bak

61
Q

If BCL2 is present then what will occur

A

Inhibition of Bax/Bak - survival

62
Q

What is the effect on BIM on BCL2

A

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

63
Q

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

A

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

64
Q

How does prolonged Ras signalling lead to death

A

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

65
Q

How was the coupling device linking CytC to caspase activation discovered

A

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

66
Q

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

A

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

67
Q

Describe how genotoxic stress causes apoptosis

A 
Work through p53 
Transcription factor induces Noxa and Puma
Inhibit BCL2 
No inhibition of Bax
Caspase activation
68
Q

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

A

90%

BMS379 - Cancer Biology (11 decks)

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