Tumour Suppressor Genes

Question 1

Cell cycle

Answer 1

Unidirectional

Regulated by phosphorylation (CDK activation by cyclins)

Ubiquitination
(degradation of cyclins)

Question 2

Cell cycle control by cyclin-CDKs

Answer 2

CDK1/cyclinA = G2
CDK1/cyclinB = M
CDK4/6/CyclinD = G1
GDK2/CyclinE = G1/S
CDK2/CyclinA = S

Question 3

Oncogenes and TSGs in cell cycle

Answer 3

Activate cell cycle:
Growth factors
Oncogenes
Cyclins + CDKs

Inhibit cell cycle:
CDKi
=(CDK inhibitor)

Question 4

Dominance and recessive nature of tumourgenic phenotype

Answer 4

Normal cell + cancer cell
= hybrid cell

Lost ability to form tumours when injected into animals

.:. genes must exist in normal cells that suppress the malignant cancer phenotype

Cancer alleles = recessive

Question 5

Retinoblastoma

Answer 5

> rare childhood cancer of the eye
tumour of retina arising in precursors of photoreceptors
sporadic or heredity
1st TSG identified
even if you remove Rb in familial cases -> can still get cancers later on in life

Question 6

Tumours arise from a stem cell precursor

Answer 6

RPE cells

= retinal pigment epithelial cells

Question 7

Unilateral vs Bilateral Rb

Answer 7

Uni = 1 eye affected
- sporadic Rb
Bi = both affected
- familial Rb

Question 8

What is bilateral Rb associated with?

Answer 8

500+ fold of developing osteosarcomas during adolescence

- even if retinal tumour is removed

Question 9

Cell-Cycle dependent phosphorylation of Rb

Answer 9

1. Cells pass through M/G1
   -> Rb unphosphorylated
2. Cells progress through G1
   -> 1 phosphate attached
   = pRb hyperphosphorylated
3. Pass through R point
   -> further phosphorylated by CyclinE-CDK
   = pRb hyperphosphorylated

Question 10

Rb and E2F

Answer 10

E2F = Transcription factor

• activated genes in cell cycle
• cannot do anything when bound to Rb

CDK4 activated by Cyclin D
-> triggers phosphorylation of Rb by 2 enzymatic complexes

-> E2F goes to nucleus to activate transcription of cyclins

Question 11

What happens to the cell cycle when Rb is lost?

- in terms of E2F

Answer 11

E2F always free to activate transcription

= cell cycle never repressed

Question 12

Knudson 2-hit hypothesis

Answer 12

Familial Rb
= inherit 1 mutant Rb allele
-> 1 somatic mutation required for disease to occur

Sporadic
= 2 somatic mutations required for disease to occur

Question 13

What does the Knudson 2-hit hypothesis about the type of allele that Rb is?

Answer 13

Rb alleles are recessive
- both copies must be lost to lose Rb function

True for most TSGs

Question 14

Elimination of wild-type Rb gene copies

Answer 14

1st Rb gene copy inactivated by mutation
= heterozygous cell
= cell exhibits wild-type phenotype

2nd mutation
-> both copies inactivated
= homozygous cell
(could occur during mitotic recombination)

Question 15

What are the frequencies of mutational events in the 1st and 2nd mutations?

Answer 15

1st = 10^-6 per cell event

2nd = 10^-5 to 10^-4 per cell generation
= easier to achieve

Question 16

Gene conversion

Answer 16

1. DNA pol starts replication on red chromosome
2. jumps to homologous, green chromosome
3. copies segment of chromosome
4. jumps back to red chromosome + continues copying

Question 17

How can gene conversion result in loss of heterozygosity (LOH)?

Answer 17

DNA pol jumps between chromosomes

-> jumps to copy mutated Rb (familial cases)

= 2 copies of mutated gene

Question 18

Chromosomal non-disjunction + LOH

Answer 18

In mitosis
- by chance end up with 1:3 ratio of chromosomes between 2 daughter cells

• > 1 cell ends up with 3 chromosomes - 2 mutated chromosomes
• > loss of normal chromosome

Question 19

Chromosomal localisation of Rb locus

Answer 19

Karyotyping of Rb showed loss of 1000s of kilo bases:

Rb genes on chromosome 13
- q arm, position 14

Question 20

Detection of LOH

Answer 20

Esterase D also found at 13q14

• protein with 2 different isoforms
• represented in human gene pool by 2 alleles whose proteins migrate differently in gel electrophoresis

Question 21

Mutations of the Rb gene

- probes

Answer 21

Using Rb probes

-> showed osteosarcoma cancer cells also carried defective Rb alleles

Question 22

RFLP

Answer 22

= restriction fragment length polymorphisms

Question 23

RFLPs uses

Answer 23

To establish whether you have 2 copies of TSG

Question 24

p53

Answer 24

= ‘guardian of genome’

• 1 of most important TSGs
• controls DNA repair
• most cancer cells have mutation in it

Question 25

Epigenetics

Answer 25

Change in gene expression w/out affecting DNA sequence itself

No mutations or removal of DNA

Question 26

Epigenetic silencing of TSGs

Answer 26

Histone modification
- molecules attach to histone tails
= compacts or loosens DNA

DNA methylation
= addition of methyl groups to some bases can inactivate gene

Question 27

Promoter hypermethylation inactivates TSG

Answer 27

e.g. RASSF1A
CpG islands in promoter region 
- methylated 
-> prevents recruitment of TFs
= gene not expressed