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BMS379 - Cancer Biology > L3 - Evading Growth Supression > Flashcards

L3 - Evading Growth Supression Flashcards

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1
Q

What underpins all of the mechanism of growth supression

A

The cell cycle clock

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2
Q

Where do most growth supression mechanisms act

A

At the restriction (R) point

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3
Q

Where is the R point

A

At the end of G1 phase

This is where the cell must commit to division

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4
Q

Cells spend most of their tiime in what phase

A

G1

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5
Q

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

A

Cell imagine

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6
Q

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

A

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

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7
Q

What doesn’t cell imagine solve

A

The G1/G2 probelm

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8
Q

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

A

DNA has be replicated in S phase

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

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9
Q

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

A

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

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10
Q

Describe how FACS is occuring

A

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

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11
Q

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

A

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

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12
Q

Describe the three dimensions used in £D FACS analysis

A

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

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13
Q

Describe how the rate of DNA synthesis is measured

A

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

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14
Q

What is an advantage of 3D FACS

A

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

Allows us to identify detailed mechanisms of cell cycle arrest

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15
Q

When is G1/S cyclin high

A

High during G1 - drops at the start of S phase

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16
Q

When is S cyclin high

A

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

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17
Q

When is M cyclin

A

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

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18
Q

What is the idea behind synchronising cells

What is the technique to achieved this

A

Get them all in the same phase of the cell cycle

Mitotic shake off

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19
Q

Describe how mitotic shake off would be performed

A

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

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20
Q

What is SLBP

A

Stabiliser of histone mRNA - made exclusively in S-phase

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21
Q

Replicator selection

A

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

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22
Q

When are pre rec complexes made

A

In G1 phase without DNA polymerse

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23
Q

What does replicator selection ensure

A

That DNA replication from one place only happens once

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24
Q

Describe how repilicator selection only happens once

A

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

25
Q

The action of Cdt1 ensure

A

Cell cycle directionality

26
Q

What is geminin

A

Mops up any remaining Cdt1 to prevent rereplication

Expressed at late S/early G2

27
Q

Describe the patterns in expression of Cdt1, SLBP and geminin

A

Cdt1 - expressed first then SLBP and then geminin

28
Q

When is SLBP needed

A

During late G2

Histone H1 needs to be made to allow the reorganisation of chromosomes early in mitosis

29
Q

Why is 4 colour fluourscence hard

A

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

30
Q

G1 marker
S phase marker
G2 marker
M markers

A

g1 Cdt1
S SLBP
G2 geminin
M histone H1

31
Q

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

A

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

32
Q

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

A

Cancer phenotype is recessive

33
Q

What experiments did Rao and Johnson perform

A

Formation of heterokaryotons - monkey and mouse cells

34
Q

What is a heterokaryon

A

Cell with multiple nuceli

35
Q

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

A

No tumour was seen sicne the cancer phenotype is recessive

GENETIC RESCUE - absence of the TSGs recovered by the presence of the wildtype alleles

36
Q

What is more likely gain or loss of function mutations

A

Loss

37
Q

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

A

YES - loss of function is more likely to occur than gain of function

NO - would need to lose both alleles - this is highly unlikely

38
Q

Retinoblastoma
Morphology
Treatment

A

Whitening of the iris - can lead to blindness

Treatment involves the removal of the eye

39
Q

Two forms of retinoblastoma

A

Sporadic and famililal

40
Q

Sporadic is usually

A

Unilateral

41
Q

Familial is almost always

A

Bilateral

42
Q

Describe the incidence of non-retinal tumours in retinoblastoma patients

A

Increased

At 50 y/o

Bilateral patients have a 36% likelihood
Unilateral have a 5.7% likelihood

43
Q

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

A

For famililar - straight line

For unilateral - curve

44
Q

What did the straight line for bilateral indicated

A

FIRST ORDER REACTION

Suggests that there is one thing involved - one thing needs to go wrong

45
Q

What did the curve line for unilateral indicated

A 
SECOND ORDER RATE 
Two things (two reactants) need to go wrong for cancer to occur
46
Q

Describe the two hit hypothesis for familial Rb

A

Inherit the mutant Rb

Mutation then must occur in the paralogue

47
Q

Describe the two hit hypothesis for sporadid Rb

A

Acquire the first Rb mutation

THEN have to also acquire a second mutation

48
Q

Describe how this second mutation can occur so frequently

A

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

49
Q

What chromosome is implicated in retinoblastoma

A

Chromsome 13

50
Q

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

A

Zymography

51
Q

Describe the technique of zymography

A

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

52
Q

What is seen when running zymography of a tumour tissue

A

People never have both of the isoforms in the tumour tissue

Heterozygosity is always lost first

53
Q

What does the Rb protein interact with

A

E2F

54
Q

What is E2F

What genes are under its control

A

Principle transcription factor

Drives transcription of all genes responsible for S phase

55
Q

Describe E2f in the absence of mitogens

A

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

56
Q

Describe E2F in the presence of mitogens

A

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

57
Q

Describe the MAIN effect on E2F genes of mutant Rb

A

E2F target genes are ALWAYS on

58
Q

What is the effects of mutant Rb being present

A

Upstream E2F signalling is bypassed and now no longer relevant

BMS379 - Cancer Biology (11 decks)

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