15th Feb - Interpreting the signal

Question 1

Outline ER activation

Answer 1

ER is normally sequestered by HSP90

Oestrogen binds releasing inhibition of ER by HSP90
ER translocates to nucleus
Bind to the hormone response element
Recruits CoA which recruits HAT

Question 2

Give an example of an ER target gene

Answer 2

c-Myc

Cyclin D1

Question 3

How can ER activity be shown?

Answer 3

Reporter assay

Question 4

What is the hypoxic response?

Answer 4

HIF1 alpha is stabilised in hypoxic conditions

Question 5

What are hypoxic conditions ?

Answer 5

<1% oxygen

Question 6

How is the hypoxic response rapid?

Answer 6

HIF1 alpha is normally produced in hypoxic cells but degraded by pVHL attaching Ub

Question 7

What is the function of the hypoxic response?

Answer 7

It helps regulate oxygen homeostasis through:

• stimulating angiogenesis
• pH regulation
• promoting glucose metabolism rather than OXPHOS

Question 8

What does the hypoxic response produce?

Answer 8

VEGF

Erthyropoeitin (EPO)

Question 9

What is eryhthropoeitin (EPO)?

Answer 9

A GF which stimulates red blood cell growth

Question 10

Outline the 1D structure of p53

Answer 10

TAP1-TAP2—CoreDNA binding domain—TET–REG

Question 11

What are the main downstream genes of p53?

Answer 11

BAX
p21Waf1
GADD45/PCNA
MDM2

Question 12

What is the function of BAX?

Answer 12

It triggers the intrinsic apoptotic pathway

Question 13

What is the function of p21 waf1?

Answer 13

It is a CDK inhibitor

Question 14

What is GADD45/PCNA?

Answer 14

growth arrest and DNA damage 45/proliferating cell nuclear antigen

Question 15

Outline the mouse genetics experiments which demonstrated the p53/mdm2 loop

Answer 15

WT mice have a 2-3 year lifespan
p53 -/- mice = viable, die at 6 months from leukaemia/lymphoma
mdm2-/- mice = die as embryos due to uncontrolled p53 –> apoptosis
p53 -/-, mdm2 -/- = viable, die around 6 months

Therefore mdm2 regulates p53 levels preventing OE

Question 16

How are tumour suppressors normally inactivated?

Answer 16

Through DNA methylation

Question 17

How is HIF1 alpha signalling maintained by cancer cells?

Answer 17

Hypoxic conditions within a tumour

Loss of pVHL –> constitutice HIF1–> sustained angiogenesis

Question 18

What percentage of breast cancers are ER positive?

Answer 18

70%

Question 19

What percentage of clear cell renal cell carcinoma has pVHL suppression?

Answer 19

about 75%

Question 20

What percentage of sporadic clear cell renal cell carcinoma have biallelic inactivation of pVHL due to either mutation, deletion or hypermethylation of the promoter that releases VHL expression?

Answer 20

about 70%

Question 21

Where do most mutations occur in p53?

Answer 21

In its DBD

Question 22

What are the different ways to inactivate p53?

Answer 22

Virus
Nuclear exclusion
PTEN
MDM2
Upstream signals
Mutation

Question 23

Could p53 be reactivated?

Answer 23

Ventura 2007
Mice models with a reactiveable p53 KO allele, generated by inserting a transcription-translation stop casette flanked by lac P in vitro in the 1st intron of the endogenous WT p53 locus
For temporal control of p53 created CreER allele which targeted the ROSA26 locus
These 2 breeds of mice were crossed allowing p53 to be controlled by ER

Showed reactivation of p53 causes mass apoptosis of tumour cells