cell cycle control

Question 1

What types of tumours are associated with people that inherit an inactivated copy of RB1 ie part of the RB1 cancer syndrome?

Answer 1

Primitive neuroectodermal tumours (PNET) in the CNS
Soft tissue and osteosarcomas
Melanomas
Lung and bladder carcinomas

Question 2

What does complete loss of RB1 result in?

Answer 2

This leads to benign retinomas with fleurettes which may remain stable for life or progress to retinoblastomas showing that loss of pRb function can deregulate the cell cycle but cannot by itself generate cancer as the senescent state is activated and maintained p16 and p130 however this senescent state is associated with low level genetic instability
Escape from this senescent state requires further (epi)genetic events which induce clonal expansion and malignancy indicated by the presence of necrotic, apoptotic and mitotic cells
This malignant state is associated with high level genetic instability

Question 3

What proteins are part of the RB family?

Answer 3

These proteins control the restriction site and include pRb and the pocket proteins, p130 and p107
The function through control of the transcription factors of the E2F family and are themselves regulated through cyclin dependant kinases

Question 4

What is the function of pRb?

Answer 4

This inhibits the activating E2Fs which drive transcription from many genes involved in S phase progression

Question 5

What is the function of p130 and p107?

Answer 5

These activate the inhibitory E2Fs (E2F4,5)

Question 6

What must occur to the pocket proteins before cells can enter the cell cycle?

Answer 6

The pocket proteins must be inactivated by CDKs
Cyclin D-CDK4 phosphorylates the pocket proteins leading to their dissociation from E2F proteins and the initiation of transcription where the activator E2Fs induce transcription of their own genes and of cyclins E and A

Question 7

How do Mitogens activate CDK4 and 6?

Answer 7

Through inducing synthesis of cyclin D1, formation of cyclin D1-CDK4 complexes
There is nuclear translocation of cyclin D-CDK4 complexes
Stabilisation of cyclin D through PI3k/Akt inhibition of glycogen synthase kinase 3beta which phosphorylates cyclin D on T286 leading to nuclear export and degradation

Question 8

What occurs as the cyclin D-CDK4 dimer become abundant in cells?

Answer 8

It sequesters p21 and p27 in quiescent cells, these proteins bind to and inhibit any cyclin E-CDK2 that may be present which normally bind to an inhibit Cyclin E-CDK2
This allows CDK2-E to become active and when the p27/Cyclin E-CDK2 ratio is decreased to a critical threshold it will phosphorylate free p27 on T187 triggering its degradation

Question 9

How can cell cycle blocking occur through p27?

Answer 9

If mitogens are removed and cyclin D concentrations decrease, p16 or p15 is induced, binds to CDK4 disrupting cyclin-DCDK4-p27 complexes allowing release of p27 so it can bind to cyclin E-CDK2 leading to its inhibition

Question 10

What are the mechanisms which may lead to inactivation of the R point in cancer?

Answer 10

Genetic mutation or deletion of RB1 or RB2 (in the nuclear localisation sequences causing cytoplasmic rather than nuclear degradation)
Increased pRb degradation
MYC-induced Id2 proteins
Viral targeting of Rb proteins
Over expression of cyclin D1
Excess Cdk4
Loss of p16

Question 11

How can increased pRb degradation occur?

Answer 11

The LXCXE motif protein gankyrin is over expressed in liver cancers it increases proliferation rate, promotes anchorage independent proliferation and enhances tumorigenicitiy
It binds to pRb to increase its phosphorylation and accelerate its degradation it is a subunit of the 26S proteasome

Question 12

How do MYC-induced Id2 proteins lead to inactivation of the R point?

Answer 12

RB-/- mice die before birth with delayed differentiation of haematopoietic and neuronal cells because pRb binds and inihibits Id2 which suppresses differentiation
Neuroblatomas with amplified N-MYC oncogenes express Id2 in molar excess over pRb overcoming cell cycle arrest

Question 13

How does viral targeting of proteins deregulate the cell cycle?

Answer 13

The HHV-8 cyclin K is a variation of the cyclin theme, Cyclin K-CDK6 phosphorylates pRb and also p27kip1 on T187 to cause its debradation and activate CDK2
This unusual Cyclin K-CDK6 does not require p27kip1 for nuclear translocation unlike cyclin d-CDKs and remains active

Question 14

How can over expression of cyclin D1 lead to deregulation of the R point?

Answer 14

This may occur as a result of gene amplification or translocation. Mantle cell lymphomas possess a t(11;14) activating the cyclin D1 gene they may also lose pRb or p16 suggesting that there may be varying degrees of R point disruption

Question 15

How can excess Cdk4 activity occur?

Answer 15

Gene amplification or mutations that prevent p16 binding

Question 16

How does p16 loss lead to R point deregulation?

Answer 16

This can occur due to gene mutation or promoter hypermethylation abberations of pRb and p16 tend to occur reciprocally with gliomas tending to lose pRb or lose p16 or amplify cdk4, NSCLC generally lose p16 whereas SCLC generally lose pRb

Question 17

What may lead to G1 deregulation?

Answer 17

This may follow over-expression of cyclin E or loss of the TGFbeta pathway, mutations often occur in E2F4 in stomach and colorectal cancers deficient in mismatch repair

Question 18

What proteins does pRb bind that play a role in chromatin structure?

Answer 18

It binds to Suv39H1 and Suv420H1/2 which both generate repressive histone modifications involved in formation of heterochromatin
Histone 3 lysine 9 methylation acts as a mark to recruit heterochromatin binding protein 1
H4K20 binds proteins involved in compacting nucleosomes especially near the centromeres
There is a demethylates retinoblastoma binding protein 2 which removes methyl groups from H3K4Me3 whichi s a mark of activated chromatin
The BRM and BRG1 ATPases that are involved in nucleosome repositioning and chromatin remodelling
Cohesion and condensin II which act in gene regulation by interacting with enhancers, silencers and insulators
DNA methyltransferase which recruits HDAC, loss of pRb may lead to mislocation of this causing global hypomethylation and local hyper methylation of oncogenes

Question 19

What role does pRb play in the maintenance of genetic stability?

Answer 19

Loss of pRb may lead to chromosome instability (CIN phenotype) and the ongoing generation of aneuploidy
Some genome destabilisation events may be the result of E2F deregulation as when Mad2 (meta phase kinase which senses microtubule-kinetochore engagement) is over produced leading to defects in mitosis
Altered interactions between Suv420H1/2, cohesion and condensin may lead to mitotic abnormalities
pRb is needed for arrest in G1 following DNA damage, pRb inactivation also leads to premature S phase with replicative stress and DNA breakage