Module 2- Cell Cycle

Question 1

What needs to be regulated in the cell cycle

Answer 1

Each chromosome only duplicated once, chromosomes separates and distributed- coordinated with cell growth and only when required
Damaged cells dont replicate
G1 checkpoint/ restriction
G2 checkpoint
M checkpoint

Question 2

how many cells in body and how many of these replaced daily

Answer 2

10^13 in body with 10^10 replaced daily

Question 3

Detail on each cell cycle checkpoint- what is checked in each

Answer 3

G1= are cells ready to divide- is it big enough, is there enough energy and other resources, is DNA damaged
G2= how is the DNA- did chromosomes get correctly replicated, is DNA damaged
M checkpoint= how is chromosome alignment- did all chromosomes line up in centre of the cell ready to be pulled apart

Question 4

Where do different CDKs operate

Answer 4

4/6 with cyclinD in G1
2 with cyclinE before S
2 with cyclinA in S and start of G2
1 with cyclinB for M
7/MAT1 with cyclinH for activation of CDKs

Question 5

Overview of CDKs

Answer 5

Kinases which drive cell cycle, core enzymes of machinery
Ser/Thr protein kinases and constitutively active- activity is regulated
P >1000 proteins

Question 6

CDK structure

Answer 6

N and C lobes (B sheets and a helices respectively)
Get P for P from ATP
Helix L12 site for binding
Change from active to inactive through T loop (thr160)
Signals feed in to control activity of these enzymes

Question 7

Roles of geminin and Cdt1 in cell cycle- how was it seen

Answer 7

See with levels of them in different parts of the cell cycle
Geminin= inhibits early parts of DNA replication, switched off for G1 and on for S- also inhibits cdt1
Cdt1- helps organise replication

Question 8

Visualising the cell cycle

Answer 8

Tags on certain proteins which are expressed at different levels in different stages of the cell cycle with different colours- see cells in different stages, can look at effect of drugs or mutation also or if things arent working
Cdt1 for G1, SLBP for S, geminin for everything except G1, H1 for all DNA

Question 9

CDK regulation

Answer 9

Cyclin binding
Phosphorylation= activating or inhibitory
Dephosphorylation= activating
Binding of regulatory proteins

Question 10

What are cyclins

Answer 10

Proteins which bind to CDKs and regulate their activity- CDKs only function when cyclin bound
Expression is cyclical/ regulated- can work out which ones are needed when based on when expressed with western blot

Question 11

Cyclin structure and interaction with CDKs

Answer 11

Core helical structure= cyclin box bundles of a-helices
Helices 3 and 5 make contact with CDKs (hydrophobic interactions)
N-term is reg region- can have extra seq to control activity= more variable
6 important in humans- D1,2,3, E, A, B
In CDKs, T-loop and PSTAIRE helix move with interaction so opens up S binding site. Thr160 then P by CAK for activation

Question 12

What is CAK

Answer 12

Complex of CDK7, cyclin H and mat1
P CDK T loop at Thr160 for activation
Optimises active site conf via H bond network
Helps support ATP binding site structure
Not usually a rate-limiting step

Question 13

Regulatory phosphorylation of CDKs= limit activity to correct phase

Answer 13

Thr14 and Tyr15 regulatory P sites in roof of ATP binding site
Myt1 P both sites, Wee1 P Tyr15 and both cause inactivation as prevents peptide binding
CDC25 phsophatase family A/B/C remove inhibitory P so cause activation

Question 14

What happens when wee1 and cdc25 mutated in cells

Answer 14

Wee1 not there= get cell division but no growth as inhibits mitosis until cells are big enough
Cdc25 not there= keeps growing but no division occurs as there is no mitosis occurring

Question 15

Protein-protein interactions to control CDKs (CKIs)

Answer 15

CKI- cyclin kinase inhibitors
INK family and CIP/KIP family

Question 16

INK family of CKI

Answer 16

Inhibit CDK4/6 through binding to kinase monomer
p15, p16, p18 and p19
INK4 binds on opposite CDK side to cyclin binding, shifts N lobe of CDK so cyclin cant bind and ATP binding site also changed so ATP cant bind

Question 17

CIP/KIP family of CKI (CDK interacting protein/ kinase inhibitory protein)

Answer 17

Interact with CDK-cyclin complex
p21, p27 and p57
Eg when p27 binds, sits over top of complex and dostorts ATP binding, blocking substrate for the kinase binding. Can be P and this changes conf so it no longer inhibits

Question 18

Over view of ubiquitin mediated degradation

Answer 18

E1 uses ATP to get ub, transferred to E2 and then E3 catalyses transfer of ub onto protein
E3 provides specificity- two main types being SCF and APC/C (anaphase promoting complex)

Question 19

SCF complex relevance to G1/S phase

Answer 19

Complex with Skp1 adapter, Cullin scaffold, Rbx1 catalyst and F-box
F-box protein provides specificity eg SCFskp2 degrades CKIs and SCFfbox4 degrades cyclin D
Constitutively active, rate determined by f-box affinity for substrate and regulated by S phosphorylation- needs P for recognition by f-box

Question 20

Signals controlling entry into the cell cycle

Answer 20

Growth factors and hormones (mitogens) external factors which signal for expression of cyclin D through NGF pathway
Nutritional sufficiency internal factor which signals for expression of cyclin E and inhibition of anti-mitogenic signals through mTOR
Anti-mitogens which inhibit progression into S phase

Question 21

Regulation of transcription at G1/S phase

Answer 21

Regulates expression of proteins required for S phase eg histone unwinding, nucleotide synthesis and DNA synthesis
E2F 1-3 activators bind to DNA and E2F4-8 with p107 and p130 inhibitors bind to DNA
Rb protein binds to E2F proteins, when phosphorylated by CDK4/6 and CDK2 is inactive so releases E2F proteins
Depends where they are localised in cells too leads to regulation also

Question 22

events which occur entering the cell cycle

Answer 22

Cyclin D is expressed through ERK from external signal which activated CDK4/6
CDK4/6 P many proteins including Rb, allowing E2F1-3 to be active and bind to target genes, allowing cyclinE expression
CyclinE activates CDK2 which P proteins and hyperP Rb so it really doesnt inhibit at all
CKIs also P and degraded so they dont inhibit the process
Cross-talk and checks involved= complex

Question 23

How do anti-mitogens work eg TGF-beta

Answer 23

Inhibit progression through restriction checkpoint (G1 checkpoint)
TGF-beta acts through SMAD family of TFs
Causes expression of cyclin kinase inhibitors such as p27

Question 24

What does cancer need to do in relation to the cell cycle- how do therapeutics relate

Answer 24

Overcome G1-S checkpoint with inhibitors (Rb, CKIs) or cause inappropriate active mitogen signalling
Therapeutics aim to inhibit G1-s transition

Question 25

What is needed for cancer cells to get past the G1/S checkpoint

Answer 25

Independence of growth factors
Resistance to antimitogens

Question 26

Inhibition of Rb for controlling G1/S progression in cancer

Answer 26

Rb/E2F pathway dysregulated in nearly all cancers
Directly and indirectly

Question 27

Direct inhibition of Rb

Answer 27

Inactivating mutations
Sequestration by viral oncoproteins

Question 28

Indirect inhibition of Rb

Answer 28

CKI-resistant Cdk4 mutations
Overexpression of cyclin Ds
CKI deletion
Overactivation of mitogen signalling pathways (Her-2, Ras, c-myc)= more cyclin D
E2F overexpression

Question 29

C-myc in overactive mitogen signalling

Answer 29

Is a TF which regulates 15% of genes in genome, involved in many parts of the cell cycle and allows progression through the cell cycle
Downstream of mitogen signalling
In Burkitts lymphoma patients= cancer predisposition- 8;14 translocation where c-myc at Ig heavy chain promoter region so overexpressed

Question 30

Ras in overactive mitogen signalling

Answer 30

Activating mutations cause Ras to be permanently GTP bound state which activates MAPK pathway
Found in 25% of cancers

Question 31

Her-2 in overactivating mitogen signalling

Answer 31

Codes for human EGFR2 (GF receptor) which causes receptor to be constitutively overexpressed in breast cancers
Targeted with monoclonal antibody herceptin

Question 32

Features of palbociclib

Answer 32

Targets CDK4/6 specifically- difficult to do as CDKs all have similar structures
Dont want to target all CDKs as then healthy cells cant go through the cell cycle and die
Took a very long time before people recognised how good it was and was approved by FDA
Usually used in combination to target different areas of cancer and prevent resistance development

Question 33

Three ways palbociclib was shown to be effective/ ways drugs are shown to be effective against cancer

Answer 33

Specific effect- directly on Rb, caused less CDK4/6 so less Rb phosphorylation on Ser780 so increased Rb activity (western blot)
Broader effect- on cell cycle, stops cell cycle at G1 and prevents leaving G0/G1 (flow cytometry)
In vivo- effect on tumour growth, tumour tissue into SCID mice, higher dose caused longer time for tumour to grow and some memory effects

Question 34

Germline mutations and cancer- CDKN1C

Answer 34

Encodes KIP2, an imprinted gene (expression regulated by what one is from mother/father)
Underactivity= Beckwith-Wiedemann syndrome
Overactivity= IMAGe syndrome, Silver-Russell syndrome

Question 35

Features of Beckwith-Wiedemann syndrome

Answer 35

Under activity of CDKN1C encoding KIP2
Over growth
Ear creases, macroglossia (large tongue), hyperinsulinism, hypoglycemia (leads to obesity), abdominal wall defects, exomaphalus, cancer risk in kidneys

Question 36

Features of silver-russell syndrome

Answer 36

Germline mutation in CDKN1C which encodes KIP2
Over-activity of protein
Under growth
Facial gestait, relative microcephaly, feeding difficulties, low BMI, clinodactyly V

Question 37

CDKs and mechanics of DNA replication

Answer 37

Important for helping cells go through early stages of DNA replication
When mutated can cause reduced helicase loading (ORC and MCM helicase mutations)= Meier-Gorlin syndrome- reduced growth, facial dysmorphism, small ears and absent kneecaps
Slows cell cycle= less cells= less growth and not enough time to build stem cell reservoirs