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BMS379 - Cancer Biology > L8 - Inducing Angiogenesis > Flashcards

L8 - Inducing Angiogenesis Flashcards

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1
Q

What can be said regarding tumour vasculature

A

It is hihgly disorganised

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2
Q

Why might we expect the tissue achitecture of tumours to be disorganised

A

Due to the fact the genomes are instable

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3
Q

What is the result of disorganised vasculature

A

Hypoxia

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4
Q

Describe how SEM can allow visualisation of the vasculature How can this be quantified

A

Can allow us to tell the difference between normal and abnormal COunt, dimater, vessel size, branch poitns etc

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5
Q

What technique allows us to visualise the vasculature

A

SEM

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6
Q

Describe the process of microvascular corrosion casting

A

Introduce a fast setting polymerDissolve away all of the surounding tissues to see the vasculature left behind Coat this in an electron refractory materal to visualise under scanning electron microscopy

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7
Q

Is the absence of regularity common or rate?

A

Common

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8
Q

Describe the effect of the changes of inter vascular distance

A

There is a zone of low O2 tension

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9
Q

Describe what is seen in the region of hypoxia and anoxia

A

Reduced ATP and glucose Increase lactate levels

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10
Q

What was used for the triple stain when looking for regions of hypoxia

A

Antiepithelial - marker Anticarbonic anhydrase Antipromonidazole moiety

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11
Q

What does the anti epithelial marker (CAL-E) stain

A

Blood vessels

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12
Q

Where is carbonic anhydrase expressed

A

In regions of low O2 tension

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13
Q

What does anti primnidazole moiety stain/how does it work

A

In absence of O2 binds free sulphhydryl groups of proteins Modifies proteins and can detect in proteins Can stain

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14
Q

What happens to anti-promoidiazole moiety when O2 is present

A

Reaction to proteins is reversible - can be washed away

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15
Q

Describe staining when looking for hypoxic regions

A

Around necrotic regions - so middle of the tumour is actually dead

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16
Q

Studies into hypoxia came from what cancer

A

Renal cell carcinoma

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17
Q

What does VHL stand for

A

Von hupple lindau disease

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18
Q

Characteristics of VHL disease

A

Cerebral, spinal cord and retinal hemangioblastomas Pheochromocytomas Clear cell renal cell carcinoma

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19
Q

Penetrance of VHL by age 65

A

90%

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20
Q

Genetic inheritance of VHL

A

Autosomal dominant With tumour supressor characteristics

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21
Q

What rule does VHL follow

A

Knudsens two hit hypothesis for familial and sporadic form

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22
Q

RCC lines express high levels of

A

V-EGF

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23
Q

V-EGF is a

A

Inducer of angiogensis

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24
Q

What does V-EGF bind

A

A tyrosine kinase

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25
Q

The V-EGF pathway is

A

Autocrine

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26
Q

Paper for this lecture

A

The tumour supressor protein VHL targets hypoxia inducible factors for oxygen dependent proteolysis

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27
Q

What is the effect of VHL of V-EGF expression

A

VHL represses V-EGF

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28
Q

Describe the results of WB RCC4 (loss VHL) for VEGF and GLUT1 in normoxic and hypoxic conditions

A

Presence of VEGf in normoxic - increases in hypoxic conditions Presence of GLUT1 in normoxic increase in hypoxic conditions

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29
Q

Describe the results of WB RCC4 OE VHL for VEGF and GLUT1

A

None detected under normoxic - see a slight increase when hypoxic

30
Q

What is the effect of cycloheximideWhat is it used for

A

Blocks protein synthesis Can change the condition of the cells and observe what happens to the proteins that were previously there

31
Q

Describe what was seen when exposing RCC4 cells to VHL

A

HIF1 and HIF2 are stable over the 2 hour period

32
Q

<p>Describe the effect of exposing RCC4 cells OE VHL to cycloheximide</p>

A

HIF1a and HIF2a fade much more quickly

33
Q

What is the conclusion regarding the experiments with cycloheximinde

A

Shows that V-EGF transcription is regulated by HIF genes and that VHL induces the destruction of HIF genes

34
Q

What was the next step following the cycloheximide expts

A

Perform expts to determine if this is a ubiquitin dependent process

35
Q

Describe the expts performed to determine if the degradation was ubiquitin dependent

A

Take lysate and add HIF1a and add recombinant VHL in presence or absence of ubiquitin

36
Q

Results of the ubiquitin study

A

Heavier band in the presence of ubiquitin showing HIF1a is being ubiquitinated
Stronger in the presence of VHL

37
Q

Conclusion from the ubiquitin study

A

Suggests VHL is inducing ubiquitination of HIF1a and in the presence of VHL this doesn’t happen

38
Q

Why are co immunoprecipitation studies performed

A

To test for an interaction

39
Q

What was the hypothessided interaction in this paper

A

Interaction between HIF1a and VHL

40
Q

Results from the coimmunoprecipitation

A

Unpredicatble

Interaction between HIF1a and VHL was dependent on where the HIF1a was sourced from

41
Q

Results from mass spec

A

In WT - looking at VHL interacting domain of HIF1a - see two peaks 16 (O) apart
Suggests difference caused by oxidation

Sequence analysis showed that the only AA that could be oxidised was a proline
When mutate to an alanine no longer the two peaks

42
Q

Describe the conformational test to see if proline was being oxidised

A

Hydrolyse HIF1a in strong acid and then run THIN LAYER CHROMATOGRAPHY

See spot for both proline and hydroxyproline

43
Q

Only ___________ containing_______ binds ________

A

Hydroxyproline containing HIF1a binds VHL

44
Q

Describe what happens at normal oxygen tension

A 
Sufficient O2 for proline hydroxylase 
Hydroxylation of two prolines in VHL binding site 
VHL able to bind 
Recruits a ubiquitin ligase 
Polyubiquitnation of HIF1a 
Proteasomal deg
45
Q

Describe what happens under hypoxia

A 
No O2 for proline hydroxylase 
HIF1a not hydroxylated 
PVL unable to bind 
HIF1a binds with HIF1b to VEGF target gene 
VEGF expression
46
Q

Angiogenesis induced in a ______ dependent manner

A

VEGF

47
Q

Describe the experiments showing that angiogenesis occurs in a VEGF dependent manner

A

Small molecule inhibitor to VEGF
Intrdoduce tumour
When inhibitor present signigicant decrease in number of vessles (dose dependent)

48
Q

Small molecule inhibitor to VEGF

A

ZD6474

49
Q

Describe the RIP TAG mouse

A

SV40 large T antigen under the control of the RAT insulin promoter

So Large T is only expressed in pancreatic islets in response to insulin

50
Q

What is the effect of large T

A

Interacts negatively with p53 and Rb

51
Q

Does hyper plasia correlate with angiogenesis

Provide the evidence

A

NO

Start to see islets growing at around 5 weeks
Hyperplastic emerge at aroud week 5 - 50%
But only 10% angiogenic after 7 weeks

HYPERPLASTIC GROWTH DOES NOT CORRELATE WITH ANGIOGENESIS

52
Q

Describe the in vitro assay performed to identify the angiogenic switch

A

Isolated endothelial cells from bovine source
Develop into vascular cells

Take islets from RIPTAG mice

Attempt to recapitulate observations in vitro

53
Q

Hypothesis for the switch

A

Switch is a swtich from -VEGF to +VEGF

54
Q

What can be said of size change of the islets between weeks 5-12

A

No net size change
Clearly balance between proliferation and apoptosis

But after this quite signigicant change in size

55
Q

Where temporally is the angio switch likely to be

A

Between when islets undergo the significant size change

Would hypothesise before increasing in size they are -VEGF and after they are +VEGF

56
Q

Effects of using a VEGF inhibitor

A

Significantly blocks the emergence of angiogenic islets and tumours at all stages in the RIP TAG mice

57
Q

SU5416

A

VEGF inhibitor

58
Q

What does IHC show for ….

VEGF

VEGFR

VEGF bound to VEGFR

A

VEGF expressed in nonangio with receptor

NO LIGAND RECEPTOR INTERACTIONS OCCURING

Ligand receptor interactions are able to occur in the tumours

59
Q

Conclusions of the IHC of VEGF/R/interactions

A

No shortage of VEG-F in islets

Just no engagements with the receptor

60
Q

Describe the process of zymography

A

Introduce a substraye into polyacrylamide gel
Run get
Incubate - substrate used at that location

61
Q

Describe how zymography was used to investigate effects of VEGF

What were the results

How did this change the hypothesis

A

Embed a gelatin (ECM) protein and then look for enzymes capable of degrading

See when the angiogenic switch occurs have bands for proMMP9 and MMP9

VEGF is embedded in the membrane - MMP9 required to release it

62
Q

Do you get angiogenesis in the following situations ….

Untreated islets

Without endothelial cells

MMP2 treated

MMP9i treated

Treated with anti-VEGF

Treated with MMP9

A

No in all except when treated with MMP9

63
Q

So VEGF is made but …..

What has to occur for VEGF to interact with its receptor

A

MAde but retained by the ECM

ECM must be broken down by MMP9 to release VEGF so it is able to interact with its receptor

64
Q

What is KIT

A

RTK - oncogene

65
Q

What is seen in the absence of KIT

A

Mouse - incomplete haematopoesis

66
Q

Describe the model system used to look at where the MMP9 comes from

A

Kit KO cross with a RIP TAG

67
Q

What is seen in Kit KO cross with a RIP TAG mice

A

See only a small number of hyperplastic islets since there is NO ANGIOGENIC SWITCH

68
Q

Why is there no ANGIOGENIC SWITCH in the KIT/RIP TAG mice

A

since there are no mast cells or macrophages

69
Q

How does the angiogenic switch require mast cells and macrophages?

A

Secretion of pro inflammatory cytokines which then recui mast cells and macrophages - this is CRITICAL

70
Q

What do the mast cells secrete

What is the effect of this

A

Secretion of MMP9

Degradation of the ECM

VEGF released - this promotes angiogenesis

BMS379 - Cancer Biology (11 decks)

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