Hall 22 - Cell, Tissue, Tumor Kinetics Flashcards
Are Cdk levels constant throughout the cell cycle? What regulates their activity?
Yes
Cyclins
Cdc25 phosphatases
Cdk inhibitors
What cyclins are synthesized during G1>S?
Cyclin D + Cdk4/6
Cyclin E + Cdk2
What cyclins are synthesized during S>G2?
Cyclin A + Cdk2
What cyclins are synthesized during G2>M?
Cyclins B/A + Cdk1
What does Cdc25A do?
Removes inhibitory phosphate groups from Cdk4/6 and Cdk2 (G1/S)
Positively regulates Cdk activity (increases)
What does Cdc25C do?
Removes inhibitory phosphate groups from Cdk1 (G2)
Positively regulates Cdk activity (increases)
What inhibits Cdc25?
ATM/ATR
Phosphorylates and activates checkpoint kinases Chk1/2, which phosphorylate Cdc25 and target them for degradation
What two families inhibit Cdk?
INK4 (p16-INK4A)
Cip/Kip (p21)
How do INK4 family proteins inhibit Cdk?
p16-INK4A boosts Rb
p14-ARF inactivates MDM2 (boosts p53)
How do Cip/Kip family proteins inhibit Cdk?
p21 (Cip1) is a key p53 target gene
What is BMI1?
An oncogenic polycomb protein that is a negative regulator of INK4A/B
How does TGF-beta inhibit Cdks?
Inhibits Cdc25A activity (via Smad3/4) and increases INKs and p21
How does growth factor withdrawal inhibit Cdks?
Activates GSK-3beta > phosphorylates cyclin D and targets for degradation
What checkpoint is lost in cancer cells?
G1>S checkpoint (loss of p53 function)
Retain the intra-S and G2 to M checkpoints
What does Rb do at rest?
Inhibits E2F/DP-1 transcription factors
When Rb is phosphorylated by Cyclin D-Cdk4/6 and Cyclin E-Cdk2, the phospho-Rb and HDAC repressor complex dissociate from DNA > increase E2F/DP-1
What happens when Rb is phosphorylated?
When Rb is phosphorylated by Cyclin D-Cdk4/6 and Cyclin E-Cdk2, the phospho-Rb and HDAC repressor complex dissociate from DNA > increase E2F/DP-1
E2F drives transcription of key genes for cell cycle progression and DNA replication (including more Cyclin E = positive feedback loop)
How is the G1 to S checkpoint regulated?
DNA damage > ATM auto-phosphorylates (inactive dimer > active monomer)
> phosphorylates p53 and MDM2 to release p53 inhibition > p53 drives transcription of p21 > inhibits Cdk4/6 and Cdk2
ATM phosphorylates Chk1/2 > inhibits Cdc25A > decreased Cdk4/6 and Ckd2
What does p53 do?
Regulates G1 to S checkpoint
ATM monomer > phosphorylates p53 and MDM2 to release p53 inhibition > p53 drives transcription of p21 > inhibits Cdk4/6 and Cdk2
ATM phosphorylates Chk1/2 > inhibits Cdc25A > decreased Cdk4/6 and Ckd2
How does HPV abrogate the G1 to S checkpoint?
Inhibits both p53 and Rb with E6 and E7 viral proteins (respectively)
What is the most variable length phase in the cell cycle?
G1
What are four ways that tumors inactivate p53?
- Viral proteins: E6 (HPV), adenovirus E1B, SV40 T antigen
- Point mutations in p53
- MDM2 amplification (destabilizes p53 for ubiquitination)
- Loss of INK4A-ARF locus (less p14-ARF = increased MDM2 activity)
When is Cyclin A maximally expressed?
S phase
Forms Cyclin A-Cdk2 complex
What is Cdc45?
Crucial to loading DNA polymerase alpha onto chromatin and for replicon initiation (S phase)
How does ATM act in intra-S phase checkpoint?
Phosphorylates Chk1/2 > inhibits Cdc25A > decreased Cdk2 activity
Phosphorylates NBS (in MRN) > activates SMC proteins > intra-S arrest
*If ATM or NBS is deficient, then cells have radioresistant DNA synthesis
What drives the G2 to M transition?
Cyclin B-Cdk1
What inhibits Cdk1?
Wee1 and Myt1
What activates Cdk1?
Aurora A kinase (AurA)/Bora activates PLK1 > activates Cdc25C > activates Cdk1
How does ATM act in G2 to M checkpoint?
Phosphorylates p53 (relieves MDM2 inhibition) > key target genes transcribed:
- 14-3-3 sigma
- GADD45
- p21 (inhibits Cdk1)
- Pro-apoptotic genes (Bax, Noxa, Puma)
- PCNA
- MDM2 (negative feedback loop)
What co-activator complex acetylates and increases p53 in G2 to M?
p300/PCAF
What does ATM phosphorylation of Chk1/2 in G2 to M do?
Inhibits Cdc25C > decreased Cdk1
Chk1/2 also phosphorylates p53, increasing activity, and Wee1
How does ATR sense DNA damage?
Senses persistent single-stranded DNA (stalled replication forks, DSB breaks after resection of one DNA strand)
Recruited by ATRIP
Targets Wee1, Chk1 > cell cycle arrest
What is the major protein involved in late G2?
ATR
*Not ATM (which is the major protein involved in response to DNA damage overall)
Regulates firing of replication origins, repair of damaged replication forks, and preventing premature mitosis
What cell cycle checkpoint does caffeine inhibit?
G2 to M
(more likely to proceed to mitosis)
What is the most important cell cycle checkpoint after radiation damage?
G2 to M
1 Gy in G2 = 1 hour delay in cell cycle progression
Deficient G2 checkpoint arrest = radiosensitivity (unrepaired DNA damage going into mitosis)
What is the mitotic index?
Proportion of cells in mitosis at any given time
MI = λ x TM / TC
λ = 0.693, correction factor for cells doubling during mitosis
TM = length of mitosis
TC = total length of cell cycle
(provides ratio of M relative to total cell cycle length, but not absolute duration)
What is the labeling index?
Proportion of cells in S phase at any given time
LI = λ x TS / TC
λ = 0.693
TS = length of S phase
TC = total length of cell cycle
(provides ratio of S relative to total cell cycle length, but not absolute duration)
What technique can give the absolute duration of all cell cycle phases?
Percent labeled mitoses technique
Flash label cells and harvest/stain at periodic intervals, plot M percentage vs time
What is the average cell cycle time TC?
48 hours
TS = 16 hours
~TC = 3 x TS
What is the growth fraction?
Fraction of cells proliferating at any given moment
GF = P / (P+Q)
P = proliferating cells
Q = quiescent cells
GF = 30-50% in most solid tumors
What is the potential doubling time?
Tpot = cell doubling time of a tumor assuming no cell loss
Tpot = TC / GF = λ x TS / LI
What accounts for the difference between Tpot and actual tumor doubling time (Td)?
Cell loss
Cell loss factor φ = 1 - Tpot/Td
φ = ratio of rate of cell loss vs rate of new cell production; ~77%
How are Tpot and Td related for smaller tumors?
Closer together
Td>Tpot for large tumors
What is the most important factor determining tumor growth?
Cell loss
Explains why cell cycle time is short (TC ~1-5 days) but tumor doubling time is 2-3 months
How does the cell loss factor φ change with radiation?
Decreases several weeks into radiation (rate of tumor regression slows)
Summarize tumor growth in terms of GF and cell loss.
A minority of cells (GF) are proliferating (most are quiescent)
Most new cells produced by mitosis are lost from the tumor (inadequate O2, apoptosis, immune attack, metastasis)
