lecture 16- cell checkpoints 2

Question 1

what is MYC and how does it contribute to cancer

Answer 1

transcription factor part of bHLH family which forms dimers and associate with gene promoters

high levels = oncogene

Question 2

what is the effect of the myc-max complex

Answer 2

promotes proliferation and inhibits differentiation

Question 3

what is the effect of the Mad-max complex

Answer 3

inhibits proliferation and promotes differentiation

Question 4

what actions do myc-max have on the cell clock

Answer 4

increased cyclin D2/CDK4
pRb hypophosphorylation
E2F trapped

Question 5

what actions do myc-miz1 have on the cell cycle

Answer 5

decreased CKI transcription
increased cyclin E/CDK2
increased p27kip1 degregation

Question 6

how was it shown that MYC acting alone can relieve all contraints on proliferation

Answer 6

engineered cells to express a chimeric Myc–estrogen receptor (Myc-ER) fusion protein.

Myc is inactive until 4-OHT is added.

4-OHT binds to the ER domain, causing Myc to translocate to the nucleus and become active—in a controllable, inducible way.

Cells deprived of growth factors (in G0) stayed non-dividing unless stimulated.
But when 4-OHT was added, activating Myc-ER, cells entered the cell cycle—without any growth factors.
This showed that Myc activation alone was sufficient to drive proliferation.

Question 7

how can Myc be targetted during cancer

Answer 7

direct inhibitors block dimerisation of MYC/MAX or interfere with binding of dimers to DNA

indirect inhibitors interfere with MYC at the transcriptional level

Question 8

describe the experiment to show how myc inhibition results in tumour regression in pancreatic cancer (Cre/LoxP system)

Answer 8

made transgenic mice with pdx1 promoter and cre

these initiate constituative expression of tTA
this induces Teto which is paired to either c-myc / GFP/ luciferase reporters

all of these can be inhibited by DOX (because DOX inhibits tTA)

results= in mice with DOX, tumours reduced dramatically

Question 9

what is mycro3 and what was the experiment to show it worked

Answer 9

small molecule inhibitor of MYC/MAX dimerisation

test= expressed 18F-FDG (labelled in glucose)
tumour cells have higher uptake of glucose so glow brighter

but when treated with mycro3 they shrinked

Question 10

how was mycro3 tested in humans and what was the result

Answer 10

orthotopic= inject human PDAC cells into pancreas of immunosupressant mouse

heterotopic= subcutaneous injection of PDAC cells into immunosuppressant mouse

result =significant reduction in growth

Question 11

How are TGFbeta and myc related

Answer 11

TBF beta counteracts myc activity

how= no binding of myc to ck1
ck1 expressed so CDK4/6 inhibited
pRb not phosphorylated
cells dont go through R point

Question 12

what happens to TGFbeta control of myc in tumours

Answer 12

myc isnt under TGFbeta control anymore

Question 13

How do cancers inactivate TGFbeta signalling

Answer 13

inactivate SMAD 2/3/4
reduce TGFbeta receptor activity

Question 14

what do very high levels of TGFbeta lead to and how does this happen

Answer 14

turns it from a tumour suppressor into a tumour promoter

occurs in later cancer stages, when cancer cells quire the ability to increase cell proliferation when stimulated with TGFbeta

Question 15

how does tgf beta help cancer cells grow in the later stages of cancer

Answer 15

helps the epithelial to mesenchymal transition

Question 16

why does inhibition of the tgf beta pathway have to be done at a specific stage in patients

Answer 16

to make sure they are catching the tgf beta when is is a tumour promotor and not a tumour suppressor

Question 17

how can we target TGFbeta (tumour promoter) signalling

Answer 17

antisense olgionucleotides
antibodies blocking receptor and ligand
receptor kinase inhibitors

Question 18

what are the dangers of targetting tgf signalling

Answer 18

inhibits all tgfbeta-induced signalling effects regardless of context

Question 19

what happens when p53 is mutated

Answer 19

it cant bind DNA

Question 20

what is the mechanism for p53 when DNA damage is detected

Answer 20

p53 phosphorylated

blocks Mdm2 via ATM phosphorylating and therefore inactivating it

so rapid increase in p53 levels

post translational stabilisation

Question 21

what happens to p53 in absence of stress or when growth factors are present

Answer 21

p53 is ubiquinated by activated MDM2 and degraded by proteosome

Question 22

what is p53s normal role in the cell cycle

Answer 22

increases p21cip1 levels
decreases CDK/cyclin levels
cell arrest

Question 23

what happens to p53 in cancer

Answer 23

cante bind DNA
decreased p21cip1
no blockage of CDK/cyclin
damaged mutations passed on through cell cycle

Question 24

what is the direct inhibitor of MYC

Answer 24

Mycro3

Question 25

what is the indirect inhibitor of MYC

Answer 25

DOX

Question 26

describe the Cre-LoxP system

Answer 26

Cre recombinase = enzyme that cuts DNA at specific loxP sites The system enables precise DNA editing lox P in.. Same direction=Deletion=Knock out genes Opposite direction=Inversion =Flip gene orientation Different chromosomes =Translocation=Rearrange DNA between chromosomes

Question 27

why is tumour growth reduced more in subcutaneous injection of drug vs. pancreatic injection

Answer 27

pancreatic cancer cells are very dense, so less drug can actually reach them subcutaneous drugs are distributed slowly

Question 28

how do we know that p53 is regulated at the level of post transcription

Answer 28

there is no difference in mRNA levels when p53 levels increase