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BMS379 - Cancer Biology > L8 - Inducing Angiogenesis > Flashcards

L8 - Inducing Angiogenesis Flashcards

(70 cards)

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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
The V-EGF pathway is
Autocrine
26
Paper for this lecture
The tumour supressor protein VHL targets hypoxia inducible factors for oxygen dependent proteolysis
27
What is the effect of VHL of V-EGF expression
VHL represses V-EGF
28
Describe the results of WB RCC4 (loss VHL) for VEGF and GLUT1 in normoxic and hypoxic conditions
Presence of VEGf in normoxic - increases in hypoxic conditions Presence of GLUT1 in normoxic increase in hypoxic conditions
29
Describe the results of WB RCC4 OE VHL for VEGF and GLUT1
None detected under normoxic - see a slight increase when hypoxic
30
What is the effect of cycloheximideWhat is it used for
Blocks protein synthesis Can change the condition of the cells and observe what happens to the proteins that were previously there
31
Describe what was seen when exposing RCC4 cells to VHL
HIF1 and HIF2 are stable over the 2 hour period
32

Describe the effect of exposing RCC4 cells OE VHL to cycloheximide

HIF1a and HIF2a fade much more quickly
33
What is the conclusion regarding the experiments with cycloheximinde
Shows that V-EGF transcription is regulated by HIF genes and that VHL induces the destruction of HIF genes
34
What was the next step following the cycloheximide expts
Perform expts to determine if this is a ubiquitin dependent process
35
Describe the expts performed to determine if the degradation was ubiquitin dependent
Take lysate and add HIF1a and add recombinant VHL in presence or absence of ubiquitin
36
Results of the ubiquitin study
Heavier band in the presence of ubiquitin showing HIF1a is being ubiquitinated Stronger in the presence of VHL
37
Conclusion from the ubiquitin study
Suggests VHL is inducing ubiquitination of HIF1a and in the presence of VHL this doesn't happen
38
Why are co immunoprecipitation studies performed
To test for an interaction
39
What was the hypothessided interaction in this paper
Interaction between HIF1a and VHL
40
Results from the coimmunoprecipitation
Unpredicatble | Interaction between HIF1a and VHL was dependent on where the HIF1a was sourced from
41
Results from mass spec
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
Describe the conformational test to see if proline was being oxidised
Hydrolyse HIF1a in strong acid and then run THIN LAYER CHROMATOGRAPHY See spot for both proline and hydroxyproline
43
Only ___________ containing_______ binds ________
Hydroxyproline containing HIF1a binds VHL
44
Describe what happens at normal oxygen tension
``` 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
Describe what happens under hypoxia
``` No O2 for proline hydroxylase HIF1a not hydroxylated PVL unable to bind HIF1a binds with HIF1b to VEGF target gene VEGF expression ```
46
Angiogenesis induced in a ______ dependent manner
VEGF
47
Describe the experiments showing that angiogenesis occurs in a VEGF dependent manner
Small molecule inhibitor to VEGF Intrdoduce tumour When inhibitor present signigicant decrease in number of vessles (dose dependent)
48
Small molecule inhibitor to VEGF
ZD6474
49
Describe the RIP TAG mouse
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
What is the effect of large T
Interacts negatively with p53 and Rb
51
Does hyper plasia correlate with angiogenesis Provide the evidence
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
Describe the in vitro assay performed to identify the angiogenic switch
Isolated endothelial cells from bovine source Develop into vascular cells Take islets from RIPTAG mice Attempt to recapitulate observations in vitro
53
Hypothesis for the switch
Switch is a swtich from -VEGF to +VEGF
54
What can be said of size change of the islets between weeks 5-12
No net size change Clearly balance between proliferation and apoptosis But after this quite signigicant change in size
55
Where temporally is the angio switch likely to be
Between when islets undergo the significant size change Would hypothesise before increasing in size they are -VEGF and after they are +VEGF
56
Effects of using a VEGF inhibitor
Significantly blocks the emergence of angiogenic islets and tumours at all stages in the RIP TAG mice
57
SU5416
VEGF inhibitor
58
What does IHC show for …. VEGF VEGFR VEGF bound to VEGFR
VEGF expressed in nonangio with receptor NO LIGAND RECEPTOR INTERACTIONS OCCURING Ligand receptor interactions are able to occur in the tumours
59
Conclusions of the IHC of VEGF/R/interactions
No shortage of VEG-F in islets | Just no engagements with the receptor
60
Describe the process of zymography
Introduce a substraye into polyacrylamide gel Run get Incubate - substrate used at that location
61
Describe how zymography was used to investigate effects of VEGF What were the results How did this change the hypothesis
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
Do you get angiogenesis in the following situations …. Untreated islets Without endothelial cells MMP2 treated MMP9i treated Treated with anti-VEGF Treated with MMP9
No in all except when treated with MMP9
63
So VEGF is made but ….. What has to occur for VEGF to interact with its receptor
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
What is KIT
RTK - oncogene
65
What is seen in the absence of KIT
Mouse - incomplete haematopoesis
66
Describe the model system used to look at where the MMP9 comes from
Kit KO cross with a RIP TAG
67
What is seen in Kit KO cross with a RIP TAG mice
See only a small number of hyperplastic islets since there is NO ANGIOGENIC SWITCH
68
Why is there no ANGIOGENIC SWITCH in the KIT/RIP TAG mice
since there are no mast cells or macrophages
69
How does the angiogenic switch require mast cells and macrophages?
Secretion of pro inflammatory cytokines which then recui mast cells and macrophages - this is CRITICAL
70
What do the mast cells secrete What is the effect of this
Secretion of MMP9 Degradation of the ECM VEGF released - this promotes angiogenesis

BMS379 - Cancer Biology (11 decks)

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