Retinoblastoma Flashcards
What is retinoblastoma?
- eye tumour
- 40% hereditary with early onset with multiple tumours in both eyes
- 60% sporadic with one tumour in one eye
- Rb protein name comes from its involvement in restinoblastoma but it is found all over the body
What checkpoints through the cell cycle are there?
- G1 checkpoints check for extracellular environment and DNA damage and is most important in cancer
- S + G2 checkpoints check for incompletely replicated DNA
- M checkpoint checks for correct chromosome attachment to mitotic spindles
- once past G a cell is commited to the cell cycle unless killed
What can cause Rb to hault the cell cycle?
- stresses such as hypoxia, DNA damage and destruction of the mitotic spindle
- activates phosphatases which return Rb to a hyperphosphorylated state and halt the cell cycle
What 4 ways are cyclin dependent kinases regulated?
- activated by cyclin binding
complexes are then: - deactivated by phosphorylation
- activated by dephosphorylation
- inactivated by CDK inhibitors that bind cyclin CDK complexes and inhibit their kinase actvivity
How was the exact importance of Rb shown in mice?
- Rb1 gene mutations cause embyronal death due to developmental issues
- replication still occurred in early stages showing that Rb is not essential until differentiation occurs
-Checkpoint proteins are therefore important for pause and exit of the cycle but not for the cycle itself
Name some molecular effectors of Rb activity
those involved in:
- development, mitochondrial biogenesis and chromatin modulation
- E2Fs are involved in cell cycle, cell death and cancer progrsesion
How does Rb change throughout the cell cycle
- unphosphorylated at G0
- monophosphorylated at G1
- hyperphosphorylated at the R checkpoint and beyond
- after exit from the M phase, phosphates are stripped off by protein phosphatase type 1 PP1
What was some evidence for Rb’s involvement in cancer?
- Rb is mutated or deleted in many cancers
- DNA tumour viruses recruit oncoproteins that disrupt growth by forming complexes with hypophosphorylated Rb
What are the pocket proteins?
Rb famiyl proteins
- Rb, p107 and p130
What is the structure of the pocket proteins?
- N terminus
- Large pocket contains A + B regions + the C-terminus
- small pocket describes just A and B
- E2F binds at the large pocket with most Rb interactions occuring at B
- p107 and 130 also have a kinase inhibitor site at their N terminus and a cyclin binding site between A and B
Where do other proteins bind to and interact with pocket proteins?
- in many different regions
- mDm2 binds at the large pocket
- cyclin D at the small pocket
- DNMT1 at B
Describe p107 and p130
- more similar to eachother than to Rb
- more extensive sequence homology
- shared cyclin binding and CDK inhibitory domains
Describe the phosphorylation that occurs to Rb during the cell cycle in more detail.
- cyclin D CDK6 leads to Rb monophosphorylation in early G1and it is still active
- Cyclin E CDK2 lead to Rb hypoerphosphorylation in late G1 to inactive Rb and allow cell cycle to continue
- hyperphosphorylaton can only occur after mono
How can anti-mitogenic signals affect Rb and the cell cycle?
- can return Rb to hypo or dephosphorylated active state through:
- attenuation of cyclin expression
- induction of CDK inhibitors
- direct modification of pRb by phosphatases
- cell cycle halted
Once through the restriction point of G1, how is Rb regulated?
- kept inactive by cyclins A, B and E containing complexes
- these are not sensitive to extracellular signals
- guarantees the execution of transition from S to G2 to M
What about phosphorylation of p107 and p130 pocket proteins during the cell cycle?
- p017 phosphorylated by CDK 2 from G1-G2
- p130 + CDK2 are important in G0 when cells are withdrawn from the cell cycle
- some overlap with Rb function but neither can replace Rb entirely
How does expression of pocket proteins change throughout the cell cycle?
- p130 high at G0 and decreases during active cycling
- pRb levels are constant
- p170 levels are induce at entry to G1
How can Rb be attenuated in cancer?
- deleted or mutated
- increases in Rb phosphorylation or degradation
- upstream effectors and viral proteins such as HPV E7 can inhibit all 3 pocket proteins
- all of these can lead to Rb inactivation that is seen in most/all cancers
How might an increase in Rb phosphorylatin occur in cancer?
- amplification of cyclin D
- loss of p16, p21 OR P27
- CDK mutations and amplifications
How might an increse in Rb degradation occur in cancer?
- amplification of MDM2
Brielfy, how do Rb and E2F regulate the cell cycle together?
- active Rb binds E2F and inactivates it stopping cell cycle progression
- hyperphosphorylation of Rb by cyclins and CDKs causes Rb to unbind E2F allowing it to be active
- E2F can then switch on DNA synthesis genes and the cell cycle progresses
What are the E2F proteins?
E2F 1, 2, 3a, 3b, 4 and 5 + more
- transcription factors
- DNA binding domain
- regulated by pocket proteins like Rb
How are E2F proteins activated?
- dimerisation domain allows them to form heterodimers with DP1+DP2
- when active E2F is shown in a diagram DPI1/2 is also there just not shown
How do Rb and other pocket proteins inactivate E2F proteins?
-transactivation domain at the C-terminus overlaps with the Rb binding domain
- if Rb is bound E2F cant act as a transcription factor
- E2F/DPI heterodimer is often already bound to its consensus sequence when Rb binds + can recruit HDACs to increase repression
What is the role of E2Fs 1, 2 and 3?
- activating E2Fs
- activate genes at the G1/S transition to allow cell cycle entry and progression
What is the role of E2Fs 4 + 5?
- repressive E2Fs
- repress E2F target gene expression
- translocate into the nucleus when in complex with Rb proteins during G0/G1
Why do E2F 4 + 5 only enter the nucleus when complexed with a pocket protein?
dont have their own nuclear localisation signal
What are the roles of E2Fs 6, 7 + 8?
- transcriptional repression
- have no Rb binding site, act independently of the Rb family
Besides being sequestered by Rb, how else can the E2F/DPI heterodimer be inactivated?
phosphorylation of DPI
Which E2Fs primarily interact with which pocket proteins?
- mammalian activating E2FFs with Rb
- inactvating E2Fs with p107 or 130
Describe the status of E2F/pocket protein complexes in the cell cycle
- In G0, p130 is bound to E2F 4+5
- in G1 RB binds 1,2 + 3
- When Rb is inactivated, 1, 2 + 3 can bind and switch on transcription to allow cell cycle progression
What kinds of genes are regulated by E2F?
- DNA replication genes like DNA polymerase
- CDK activity like cyclin D1
- growth promoting genes like c-myc
- mostly repressed in G0 and early G1 and activated at the G1/S boundary
How is E2F function limited to the G1/S boundary?
- cyclin A/CDK2 in teh S phase phosphorylate E2F causing its disassociation from promoters and its degradation
Describe the positive feedback loop involved at the G1/S boundary
- E2F/DPI switches on cyclin E
- leads to hyperphosphorylation of Rb
- also phosphorylated p27 leading to its degradation and stopping it from inhibiting cyclin E
What is the role of viral oncoproteins in the cell cycle?
- oncogenic viruses produce proteins that can inactivate Rb and allow infected cells to go through the cell cycle
- also need to deregulate p53 to stop apoptosis
- HPV 37 or adenovirus proteins
How do we know that the G1 restriction point is so important in cancer?
- at least 1 member of the cyclin D/CDK/p16/Rb pathway that controls it is deregulated in cancer
What cellular processes, other than E2F are affected whtn Rb is lsot?
- cell cycle exit
- response to DNA damage
- mitochondrial defects
- epigenetic changes
- many more all interconnected
What is the general role of Rb in apoptosis?
- regulates E2F function and acts to protect against apoptosis
What is the threshold model of activatine E2F?s
- contribute to a pool of E2F activity
- once this reaches a certain level is triggers proliferation or apoptosis
- too much E2F goes beyond proliferation, causes oncogenic stress and pushes the cells to apoptose
How do Rb and p53 mutations work together?
inactivating Rb alone just leads to p53 activation and cell death
there is selective pressure to reduce apoptosis so p53 pathways are also frequently seen mutated alongside Rb
How can Rb inactivation impact apoptosis?
- E2F1 overexpression leads to upregulation of ARF-p53 pathway, direct p53 phosphprylation and increased levels of BH3.
E2F1 can also inhibit survival signals though NFkB inhibtion and Bcl2 inhibition
What are the two opposing roles of E2F activation?
- can induce cell cycle progression through gene activation
- can also induce apoptosis by repressing survival genes and pathways and activating p53 and other proapoptotic genes
How might Rb be modified in cells permanently withdrawn from the cell cycle?
acetylation of pRb maintains is in a hypophosphorylated stateWh
What kinds of cells might be permenantly withdrawn from the cell cycle?
- neurons
- skeletal or cardiac muscle cells
