L15 - Checkpoint Control II

Question 1

What is anoikis?

Answer 1

Cell detachment-induced apoptosis

A self-defence strategy used to eliminate cells in inappropriate locations

Question 2

Which cells have anoikis resistance?

Answer 2

Cancer cells – have strategies to grow in the absence of anchorage and become anoikis-resistant

Question 3

What does the myc protein govern?

Answer 3

Governs decisions to proliferate or differentiate

Localised in nucleus and control a variety of cell function

Question 4

70% of human tumours overexpress 1 of 3 which members of Myc?

Answer 4

c-Myc, N-Myc or L-Myc

Question 5

Myc is a member of which family of transcription factors?

Answer 5

bHLH transcription factors

Basic DNA-binding domain followed by amino acid sequences forming a-Helix, a-Loop and second a-Helix

Question 6

How do members of the bHLH family interact?

Answer 6

Members of bHLH family form dimers and associate with gene promoters

Question 7

What does the myc-max complex do?

Answer 7

Promotes proliferation and inhibits differentiation

Question 8

What does the mad-max complex do?

Answer 8

Inhibits proliferation and promotes differentiation

Question 9

What are the 3 ways of inducing myc expression in cancer?

Answer 9

1. Gene amplification
2. Chromosomal translocation
3. Pro-virus integration

Question 10

Which complex regulates expression of key components of cell cycle clock?

Answer 10

Myc-max complex

Question 11

Which components of the cell cycle clock does the myc-max complex control?

Answer 11

Cyclin D2 and CDK4
- Elevated expression leads to pRb hypophosphorylation
E2F transcription factors
Myc-Miz1
- Repression of transcription of CKI - liberates cyclin E/CDK2 complex from inhibition
- Promotes the degradation of p27Kip1

Question 12

What impact does the myc-max complex have on progression through the R point?

Answer 12

All lead to progression through R point

Question 13

What experiments were used to show the powers of the Myc oncoprotein?

Answer 13

Myc expressed as a fusion protein with the oestrogen receptor
- Oestrogen or tamoxifen addition drives Myc translocation to the nucleus – active form
Cells in G0 in absence of growth factors
- Tamoxifen addition induces entrance into G1 and S phase
Shows myc acting on its own can relieve all of the constraints on proliferation

Question 14

How does TGFb control cell cycle progression? Which two molecules does it control?

Answer 14

It strongly increases the levels of p15INK4B
- Leads to inhibition of cyclin D-CDK4/6 complexes
- Cells can’t reach the R point
It weakly induces p21Cip1

Question 15

How does TGFb block cell cycle progression?

Answer 15

It prevents pRb phosphorylation blocking cell cycle progression
- It counteracts the activity of Myc

Question 16

How does TGFb impact on myc?

Answer 16

TGFb inhibits the expression of Myc and prevents Myc from binding CKI promoters

• CKI are expressed
• CDK4/6 and CDK2 are inhibited
• pRb is not phosphorylated
• Cells don’t go through the R point

Question 17

Elevated levels of Myc in tumour mean it is no longer under the control of?

Answer 17

TGFb

Question 18

How do cancers inactivated TGFb signalling?

Answer 18

½ pancreatic carcinoma and ¼ colon cancers have inactivated Smad 4
- Means TGFb signalling is compromised
The gene encoding its receptor is inactivated in the majority of colon cancers

Question 19

How do cancer cells counteract the deregulation of the pRB pathway by TGFb?

Answer 19

Inactivation of the Rb gene through mutation
Expression of high levels of cyclin D1
Down-regulation of INK4 proteins (CKI inhibitors) –> inappropriate inactivation of pRb
Mutations in CDK4 gene –> no longer able to bind to INK4 family of CKIs

Question 20

How is p53 linked to cancer?

Answer 20

p53 is a transcription factor regulating genes controlling cell growth and apoptosis
p53 tumour suppressor gene is the most frequently mutated gene in human cancers
- Most p53 mutations are in the DNA-binding domain
- Can no longer promote transcription of p53 target genes

Question 21

What signals cause p53 induction?

Answer 21

DNA damage and in response p53:

• Blocks cell cycle progression
• Mediates DNA repair
• Induces apoptosis

Question 22

What is seen following a rapid increase in p53 protein levels?

Answer 22

No difference in mRNA levels

Post-translational stabilisation of p53

Question 23

What happens to p53 in the absence of stress?

Answer 23

p53 is ubiquitinated by Mdm2 and degraded by the proteasome

Low p53 levels

Question 24

What happens to p53 under stress?

Answer 24

p53 stabilised and protected from degradation
Phosphorylation of p53 blocks Mdm2 binding - mediated by ATM/ATR/Chk2
ATM phosphorylates Mdm2 inactivating it
High p53 levels

Question 25

What happens to p53 when mitogenic and cell survival signals are present?

Answer 25

Phosphorylation-induced activation of Mdm2 –> p53 proteasomal degradation
Low p53 levels

Question 26

What is the main way p53 arrests the cell cycle?

Answer 26

Upregulating p21Cip1

This inactivates cyclin/CDK complexes

Question 27

How is p53 changed in cancer cells?

Answer 27

In p53 mutants in cancer – the p53 that accumulates in the cell cannot bind to DNA
- Cell cycle will progress regardless of any DNA damage