Brainscape's Knowledge GenomeTM


Top Flashcards (Certified)
All Flashcards

BMS379 - Cancer Biology > L8 - Inducing Angiogenesis > Flashcards

L8 - Inducing Angiogenesis Flashcards

1
Q

What can be said regarding tumour vasculature

A

It is hihgly disorganised

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

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

A

Due to the fact the genomes are instable

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

What is the result of disorganised vasculature

A

Hypoxia

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

What technique allows us to visualise the vasculature

A

SEM

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

Is the absence of regularity common or rate?

A

Common

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

Describe the effect of the changes of inter vascular distance

A

There is a zone of low O2 tension

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

Describe what is seen in the region of hypoxia and anoxia

A

Reduced ATP and glucose Increase lactate levels

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

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

A

Antiepithelial - marker Anticarbonic anhydrase Antipromonidazole moiety

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

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

A

Blood vessels

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

Where is carbonic anhydrase expressed

A

In regions of low O2 tension

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

What happens to anti-promoidiazole moiety when O2 is present

A

Reaction to proteins is reversible - can be washed away

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

Describe staining when looking for hypoxic regions

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

Studies into hypoxia came from what cancer

A

Renal cell carcinoma

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
17
Q

What does VHL stand for

A

Von hupple lindau disease

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
18
Q

Characteristics of VHL disease

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
19
Q

Penetrance of VHL by age 65

A

90%

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
20
Q

Genetic inheritance of VHL

A

Autosomal dominant With tumour supressor characteristics

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
21
Q

What rule does VHL follow

A

Knudsens two hit hypothesis for familial and sporadic form

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
22
Q

RCC lines express high levels of

A

V-EGF

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
23
Q

V-EGF is a

A

Inducer of angiogensis

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
24
Q

What does V-EGF bind

A

A tyrosine kinase

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
25
Q

The V-EGF pathway is

A

Autocrine

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
26
Q

Paper for this lecture

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
27
Q

What is the effect of VHL of V-EGF expression

A

VHL represses V-EGF

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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

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

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)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2025 Bold Learning Solutions. Terms and Conditions