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cancer > 3. Rb control > Flashcards

3. Rb control Flashcards

1
Q

name three DNA viruses that can give rise to cancer and the oncoprotein associated with them

A

adenovirus - E1A and E1B
simian virus 40 - T antigen
human papilloma virus - E6 and E7

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2
Q

what is the range of genome size for small DNA tumour viruses?

A

5-50kb

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3
Q

what are three properties of small DNA tumour viruses?

A
  • their genomes are small and manageable
  • their genomes are DNA and so they don’t go through an unstable RNA intermediate
  • they cause tumour formation in some species or cell types
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4
Q

what does the HPV virus give rise to?

A

cervical cancer

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5
Q

how many vaccines are there against HPV and what protection do they give?

A

there are two vaccines that give almost 100% protection

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6
Q

what do the different HPV vaccine target?

A

different subtypes of the HPV family

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7
Q

small DNA viruses are similar to retroviruses in what way?

A

they come in malignant and benign forms and this depends on the alleles or genes in the virus

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8
Q

what are the two malignant type of HPV that are targeted by the vaccines?

A

16 and 18

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9
Q

what do small DNA tumour viruses have in their genomes that makes them malignant?

A

dominant oncoproteins that when expressed in cells will cause a cancer phenotype

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10
Q

what are present in the more malignant HPVs? and what are present in the more benign?

A

more active E6 and E7 are present in the more malignant

compromised version of E6 and E7 are present in the more benign

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11
Q

what about HPV is enough to transform a cell?

A

E6 and E7 are enough to transform a cell

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12
Q

what does E7 do? and what about this process makes different HVP less malignant?

A

E7 disrupts interactions made between E2F and Rb

this process is less efficient in more benign HPV

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13
Q

what does EMSA stand for and what is it?

A

electrophoretic mobility shift assay
double stranded radiolabelled oligonucleotide sequences run on non-denaturing gel with target protein, if interaction is formed then migration is slower

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14
Q

what does high risk HPV indicate about the interaction between E2F and Rb?

A

that the disruption between E2F and Rb is important for allowing cancer to occur

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15
Q

what molecular weight is Rb?

A

105kDa

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16
Q

what type of gene is Rb?

A

tumour suppressor

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17
Q

how was Rb first isolated and what was special about this?

A

Rb was isolated by studying families that had inherited disruptions in the Rb gene causing retinal cancer, a white mass in the back of the eye
it was the first tumour suppresser isolated

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18
Q

what was also confirmed by looking at these families with disruptions in the Rb gene causing retinal cancer?

A

Knudsen’s two hit hypothesis was confirmed by genomic analysis of sporadic and familial retinal cancer

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19
Q

name two other family members of the Rb family? and how are they similar to Rb?

A

p107 and p130

they have a similar body plan and so do similar things

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20
Q

describe the general function of proteins of the Rb family

A

molecular scaffold for proteins to bind, this may turn them on/off or restrict their movement

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21
Q

how many different proteins are known to bind Rb?

A

over 100

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22
Q

how are Rb-protein interactions regulated?

A

phosphorylation

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23
Q

how can the phosphorylation of Rb be determined? and what will be seen with Rb from cels in G0/G1 and asynchronous cells?

A

Westen blot
Rb from G0/G1 will be hypophosphylated
Rb from asynchronous cells with be mainly hypophosphylated but will also have a band of hyperphosphorylated Rb

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24
Q

how many moles of phosphate are in hyperphosphorylated Rb?

A

16 moles of phosphate per mole Rb

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25
Q

what is Rb phosphorylation status during the cell cycle?

A

it is hypophosphorylated in G1

and hyperphosphorylated in S through to M phase

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26
Q

how does phosphorylation regulate Rb?

A

phosphorylation induces conformational changes the subset of proteins that Rb can interact with

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27
Q

what form of Rb binds E2F?

A

hypophosphorylated

28
Q

when Rb binds E7 what happens? and what else can have the same effect?

A

E2F is released from Rb and drives G1 to S phase progression
Rb phosphorylation leads to conformational changes in Rb meaning it no longer binds E2F

29
Q

when is E2F inactive?

A

when it is bound to Rb

30
Q

what is E2F?

A

a heterodimeric transcription factor that drives G1 to S phase progression by regulating gene expression that are essential for S phase

31
Q

name the different E2F subunits

A

E2F1-7

DP1-3

32
Q

what is required for good DNA binding of E2F?

A

E2F heterodimer

33
Q

different combination of E2F and DP are possible, what does this tell us about DNA binding specificity?

A

both E2F and DP bind DNA and contribute to binding specificity and so different combinations will lead to different binding specificity

34
Q

name five important domains of the E2F proteins

A
  • DNA binding domain
  • dimerization domain
  • transactivator domain
  • Rb binding domain
  • cyclin binding domain
35
Q

where is the Rb binding domain in E2F location and what does this mean?

A

it is located within the transactivator domain and so when Rb is bound to E2F recruitment of basal machinery is blocked

36
Q

what can E2F still do when bound to Rb?

A

it can still bind DNA just not activate transcription

37
Q

give an example of 6 genes whose expression is regulated byE2F

A
  • DNA polymerase alpha for S phase replication
  • cyclin E a major regulator of G1 to S transition
  • cyclin A
  • cdk1
  • DHFR is dihydrofolate reductase is the rate limiting enzyme in nucleotide biosynthesis
  • E2F meaning more E2F is produced
38
Q

how is E2F inactivated?

A
  • cyclin A is target gene of E2F
  • cyclin A binds E2F and recruits cdk2
    cdk2 phosphorylates DP subunit
  • this changes heterodimer conformation and so it can no longer bind DNA
  • cyclin A is no longer produced
39
Q

if the restriction point is not passed in G1 what happens?

A

cell enters G0

40
Q

what needs to occur to Rb for the restriction point to be passed? and how can this be deregulated in cancer? (2)

A

phosphorylation by cdks and release of E2F

  • if cdks are hyperactive they will constantly phosphorylate Rb and drive cell cycle progression
  • if Rb is mutated it may not be able to bind E2F
41
Q

mutations in Rb are not seen in as wider range of cancers as you would expect. when mutations do arise, what sort of cancers occur? and what does this suggest about cell cycle regulation?

A

when Rb is mutated this leads to distinct tumours: retinal cancer and occasional bone cancer may arise.
because cancer does not arise in other tissues this suggests that other tissue have ways of controlling their cell cycle when Rb is inactive, this is probably down to p130 and p107

42
Q

having a switch between hyper/hypo-phosphorylated Rb does not explain what?

A

the factor that cyclin E and A (targets of E2F) are produced at different times

43
Q

what are the 5 defined crystallisable segments of Rb?

A 
N terminal domain 
A domain 
S spacer 
B domain 
C terminal domain
44
Q

which part of Rb is not known much about? and why is this?

A

the N terminal domains

the rest of the protein seems to have obvious functions in cell cycle control

45
Q

which three segments of Rb are known as the pocket? and why it this?

A

A domain
S spacer
B domain
they are known to bind lots of proteins

46
Q

where does the majority of Rb phosphorylation occur?

A

C terminus

47
Q

name the three small DNA virus oncoproteins that are able to bind Rb pocket

A

E7, SV40 T antigen and adenovirus E1A

48
Q

what certain motif, that these small DNA virus oncoproteins along with other endogenous proteins have, binds the B domain? and what is this an example of?

A

LxCxE amino acid motif

this is an example of convergent evolution

49
Q

when Rb mutations occur in cancer where do they occur? and what does this indicate?

A

in the pocket

this indicates that the pocket domain is central to Rb function in regulation of cell cycle control

50
Q

give 2 examples of andogenous proteins that bind the B domain through LxCxE motif?

A

cyclin D

HDAC

51
Q

what is HDAC?

A

histone deacetylate deacetylates histones and condenses chromatin
removal of acetyl group from lysine restores positive charge meaning more strong interactions can be made with negative phosphate on DNA

52
Q

how does HDAC affect TF accessibility?

A

it makes it harder to TF to access DNA

53
Q

what is HAT?

A

histone acetyl transferase adds acetyl group to lysine, this removes the positive charge on lysine and loosens interactions made with negative histones

54
Q

how does HAT affect TF accessibility?

A

this makes DNA more accessible to TF

55
Q

when Rb binds E2F, E2F can still bind DNA, what else can bind Rb and what affect does this have?

A

HDAC can bind Rb and condense chromatin

this will supress gene expression in surrounding area and prevent cell cycle entry

56
Q

when Rb binds E2F, E2F can still bind DNA, what else can bind E2F and what affect does this have?

A

HAT can bind E2F and open up chromatin

this facilitates gene expression of nearby genes

57
Q

what is BRG and how does it function?

A
  • an ATP dependent chromatin remodelling factor
  • it uses ATP to slide nucleosomes along DNA
  • it can also alter packing by tightening/loosening the binding between nucleosomes and DNA
58
Q

which cyclin is the first to be expressed? and what does it activate?

A

cyclin D

this activates cdk4/6

59
Q

what does cdk4/6 phosphorylate? and what does this promote?

A
  • C terminus of Rb is phosphorylated
  • this promotes interactions between negative phosphates on CTD and lysine rich patch of B domain
  • making the S domain more accessible
60
Q

once the S domain is more accessible, what occurs?

A

cdk2/cyclin E can phosphorylate it at serine 567

61
Q

describe what is associated with Rb when a cell is in G0

  • where will this complex be?
  • and what does it do?
A

Rb binds E2F, HDAC and BRG
this complex will be localised to the nucleus by E2F
this complex supresses S phase factors

62
Q

expression of what factor is E2F independent?

A

cyclin D

63
Q

when CTD interacts with B domain, what is first to dissociate from the Rb complex? and what does this lead to?

A

HDAC

chromatin opens up and this affects gene expression of associated promoters

64
Q

why is cyclin E expressed before cyclin A?

A

cyclin E promoter is sensitive to chromatin structure and so dissociation of HDAC is enough to allow its expression. although E2F can bind this promoter there are lots of other transcription factors that can drive expression of cyclin E.
cyclin A expression requires E2F to dissociate from Rb.

65
Q

once cyclin E is expressed what occurs?

A

it phosphorylates the serine residue in the S region

this causes E2F dissociation

66
Q

what never dissociates from Rb?

A

BRG

67
Q

when E7 binds Rb what happens? and how does this compare to SW40 T antigen and adenovirus E1a?

A

there is enhanced Rb phosphorylation and degradation which leads to free E2F
they both have similar structure to HPV E7 and act in an analogous manner

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