L3 - Evading Growth Supression

Question 1

What underpins all of the mechanism of growth supression

Answer 1

The cell cycle clock

Question 2

Where do most growth supression mechanisms act

Answer 2

At the restriction (R) point

Question 3

Where is the R point

Answer 3

At the end of G1 phase

This is where the cell must commit to division

Question 4

Cells spend most of their tiime in what phase

Answer 4

G1

Question 5

What is the most simple way to detect cells in S phase and mitosis

Answer 5

Cell imagine

Question 6

What stains are used to determine if a cell is in S phase of M phase of the cell cycle

Answer 6

PCNA marker - marking sites of DNA replication - double staining with DNA
Stain for tubulin - reveals mitotic spindle - if their is one

Question 7

What doesn’t cell imagine solve

Answer 7

The G1/G2 probelm

Question 8

What is the difference between the cells genetic content in G1 and G2 phase

Answer 8

DNA has be replicated in S phase

So DNA in G2 phase has double the ammount of DNA compared to in G1 phase

Question 9

How can we sort cells into those with n and 2n of DNA

Answer 9

Using a fluorescent marker - stains the DNA - ammount of staining will depend of how much DNA there is to stain

Then use fluorescent activated cell forting to sort

Question 10

Describe how FACS is occuring

Answer 10

Measuring the amount of regular light that is being scattered - the amount of light that is being blocked is proportional to the size of the cell

Question 11

Descibe how if we know how long it takes for cells to double we can work out how long cells spend it a phase

Answer 11

Measure the proportion of cells (0.xx) and multiply this by the the total length of time for the cycle

e.g. mammalian cells double ever 22 hours and at any one time 45% are in G1

Length of G1 = 0.45 x 22 = 10hours

Question 12

Describe the three dimensions used in £D FACS analysis

Answer 12

Cell numbers
The relative ammount of DNA per cell
Rate of DNA synthesis

Question 13

Describe how the rate of DNA synthesis is measured

Answer 13

Introduce a modified nucleotide which can be incorporated during S phase
Mimics T and incorportates over 30 mins
Can detect the modification using fluorescence

RATE OF INCORPORATION IS PROPORTIONAL TO THE RATE OF DNA SYNTHESIS

Question 14

What is an advantage of 3D FACS

Answer 14

Allows us to distinguish between cells in Sphase arrest and G1 arrest

Allows us to identify detailed mechanisms of cell cycle arrest

Question 15

When is G1/S cyclin high

Answer 15

High during G1 - drops at the start of S phase

Question 16

When is S cyclin high

Answer 16

Builds up following the restriction phase falls following the entrance into M phase

Question 17

When is M cyclin

Answer 17

M cyclin builds up during G2 phase and falls after the metaphase-anaphase transition

Question 18

What is the idea behind synchronising cells

What is the technique to achieved this

Answer 18

Get them all in the same phase of the cell cycle

Mitotic shake off

Question 19

Describe how mitotic shake off would be performed

Answer 19

When in interphase cells have a flattened morphology with a diffuse microtubule network
Mitotic cells rounded with a flattened morphology - THESE ARE VULNERABLE TO SHEAR FORCE

Knock the cells off with a shear force and collect all of the mitotic cells in a new dish

Question 20

What is SLBP

Answer 20

Stabiliser of histone mRNA - made exclusively in S-phase

Question 21

Replicator selection

Answer 21

The idea that the places where replication will start is determined early - leads to the formation of pre-replicative complexes

Question 22

When are pre rec complexes made

Answer 22

In G1 phase without DNA polymerse

Question 23

What does replicator selection ensure

Answer 23

That DNA replication from one place only happens once

Question 24

Describe how repilicator selection only happens once

Answer 24

Cdt1 binds to the origins of replication and once replication begins this is proteolytically degraded

Question 25

The action of Cdt1 ensure

Answer 25

Cell cycle directionality

Question 26

What is geminin

Answer 26

Mops up any remaining Cdt1 to prevent rereplication | Expressed at late S/early G2

Question 27

Describe the patterns in expression of Cdt1, SLBP and geminin

Answer 27

Cdt1 - expressed first then SLBP and then geminin

Question 28

When is SLBP needed

Answer 28

During late G2 | Histone H1 needs to be made to allow the reorganisation of chromosomes early in mitosis

Question 29

Why is 4 colour fluourscence hard

Answer 29

Because need to have different excitation spectrum to absorption spectrum 4 times over the range of wavelengths for visible light

Question 30

G1 marker S phase marker G2 marker M markers

Answer 30

g1 Cdt1 S SLBP G2 geminin M histone H1

Question 31

How can 4 tag fluorescence be used to monitor cell cycle progression

Answer 31

Can track the markers over time - compare to the colour indicators

Question 32

What is the main statement that supports the existence of tumour supressor genes

Answer 32

Cancer phenotype is recessive

Question 33

What experiments did Rao and Johnson perform

Answer 33

Formation of heterokaryotons - monkey and mouse cells

Question 34

What is a heterokaryon

Answer 34

Cell with multiple nuceli

Question 35

What was seen when a cancerous and normal cell were fused and injected into an immunocompromised mouse

Answer 35

No tumour was seen sicne the cancer phenotype is recessive GENETIC RESCUE - absence of the TSGs recovered by the presence of the wildtype alleles

Question 36

What is more likely gain or loss of function mutations

Answer 36

Loss

Question 37

Evidence for and against the existence of tumour suppressor genes based on the likelyhood

Answer 37

YES - loss of function is more likely to occur than gain of function NO - would need to lose both alleles - this is highly unlikely

Question 38

Retinoblastoma Morphology Treatment

Answer 38

Whitening of the iris - can lead to blindness Treatment involves the removal of the eye

Question 39

Two forms of retinoblastoma

Answer 39

Sporadic and famililal

Question 40

Sporadic is usually

Answer 40

Unilateral

Question 41

Familial is almost always

Answer 41

Bilateral

Question 42

Describe the incidence of non-retinal tumours in retinoblastoma patients

Answer 42

Increased At 50 y/o Bilateral patients have a 36% likelihood Unilateral have a 5.7% likelihood

Question 43

Describe Knudsens observations when ploting percentage of cases not yet diagnosed and age in months

Answer 43

For famililar - straight line For unilateral - curve

Question 44

What did the straight line for bilateral indicated

Answer 44

FIRST ORDER REACTION | Suggests that there is one thing involved - one thing needs to go wrong

Question 45

What did the curve line for unilateral indicated

Answer 45

``` SECOND ORDER RATE Two things (two reactants) need to go wrong for cancer to occur ```

Question 46

Describe the two hit hypothesis for familial Rb

Answer 46

Inherit the mutant Rb | Mutation then must occur in the paralogue

Question 47

Describe the two hit hypothesis for sporadid Rb

Answer 47

Acquire the first Rb mutation | THEN have to also acquire a second mutation

Question 48

Describe how this second mutation can occur so frequently

Answer 48

Mitotic recombination frequent between sister chromatids Sgregation may then lead to daughter cells with two copies of the mutant gene LOSS OF HETEROZYGOSITY

Question 49

What chromosome is implicated in retinoblastoma

Answer 49

Chromsome 13

Question 50

What is the technique to determine that loss of heterozygosity has occured

Answer 50

Zymography

Question 51

Describe the technique of zymography

Answer 51

Using Desterase which has two forms - each isoform has a different molecular weight Run a PAGE and incubate the substrate and it will generate a product which is fluorescent and can be viualused

Question 52

What is seen when running zymography of a tumour tissue

Answer 52

People never have both of the isoforms in the tumour tissue | Heterozygosity is always lost first

Question 53

What does the Rb protein interact with

Answer 53

E2F

Question 54

What is E2F What genes are under its control

Answer 54

Principle transcription factor Drives transcription of all genes responsible for S phase

Question 55

Describe E2f in the absence of mitogens

Answer 55

E2F binds Rb - E2F is blocked from activating its target genes

Question 56

Describe E2F in the presence of mitogens

Answer 56

Mitogens turn on mRNA for cyclinD Turns of Cdk4/6 Phosphorylates Rb to the hypo phosphorylated state Cyclin E - Cdk2 then phosphorylates E2F to the hypephosphorylated state Causes Rb to fall off E2F and the chromatin Acetylation of S phase genes E2F genes are now able to be transcribed

Question 57

Describe the MAIN effect on E2F genes of mutant Rb

Answer 57

E2F target genes are ALWAYS on

Question 58

What is the effects of mutant Rb being present

Answer 58

Upstream E2F signalling is bypassed and now no longer relevant