Hall 22 - Cell, Tissue, Tumor Kinetics

Question 1

Are Cdk levels constant throughout the cell cycle? What regulates their activity?

Answer 1

Yes
Cyclins
Cdc25 phosphatases
Cdk inhibitors

Question 2

What cyclins are synthesized during G1>S?

Answer 2

Cyclin D + Cdk4/6
Cyclin E + Cdk2

Question 3

What cyclins are synthesized during S>G2?

Answer 3

Cyclin A + Cdk2

Question 4

What cyclins are synthesized during G2>M?

Answer 4

Cyclins B/A + Cdk1

Question 5

What does Cdc25A do?

Answer 5

Removes inhibitory phosphate groups from Cdk4/6 and Cdk2 (G1/S)

Positively regulates Cdk activity (increases)

Question 6

What does Cdc25C do?

Answer 6

Removes inhibitory phosphate groups from Cdk1 (G2)

Positively regulates Cdk activity (increases)

Question 7

What inhibits Cdc25?

Answer 7

ATM/ATR

Phosphorylates and activates checkpoint kinases Chk1/2, which phosphorylate Cdc25 and target them for degradation

Question 8

What two families inhibit Cdk?

Answer 8

INK4 (p16-INK4A)
Cip/Kip (p21)

Question 9

How do INK4 family proteins inhibit Cdk?

Answer 9

p16-INK4A boosts Rb
p14-ARF inactivates MDM2 (boosts p53)

Question 10

How do Cip/Kip family proteins inhibit Cdk?

Answer 10

p21 (Cip1) is a key p53 target gene

Question 11

What is BMI1?

Answer 11

An oncogenic polycomb protein that is a negative regulator of INK4A/B

Question 12

How does TGF-beta inhibit Cdks?

Answer 12

Inhibits Cdc25A activity (via Smad3/4) and increases INKs and p21

Question 13

How does growth factor withdrawal inhibit Cdks?

Answer 13

Activates GSK-3beta > phosphorylates cyclin D and targets for degradation

Question 14

What checkpoint is lost in cancer cells?

Answer 14

G1>S checkpoint (loss of p53 function)

Retain the intra-S and G2 to M checkpoints

Question 15

What does Rb do at rest?

Answer 15

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

Question 16

What happens when Rb is phosphorylated?

Answer 16

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)

Question 17

How is the G1 to S checkpoint regulated?

Answer 17

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

Question 18

What does p53 do?

Answer 18

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

Question 19

How does HPV abrogate the G1 to S checkpoint?

Answer 19

Inhibits both p53 and Rb with E6 and E7 viral proteins (respectively)

Question 20

What is the most variable length phase in the cell cycle?

Answer 20

G1

Question 21

What are four ways that tumors inactivate p53?

Answer 21

1. Viral proteins: E6 (HPV), adenovirus E1B, SV40 T antigen
2. Point mutations in p53
3. MDM2 amplification (destabilizes p53 for ubiquitination)
4. Loss of INK4A-ARF locus (less p14-ARF = increased MDM2 activity)

Question 22

When is Cyclin A maximally expressed?

Answer 22

S phase
Forms Cyclin A-Cdk2 complex

Question 23

What is Cdc45?

Answer 23

Crucial to loading DNA polymerase alpha onto chromatin and for replicon initiation (S phase)

Question 24

How does ATM act in intra-S phase checkpoint?

Answer 24

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

Question 25

What drives the G2 to M transition?

Answer 25

Cyclin B-Cdk1

Question 26

What inhibits Cdk1?

Answer 26

Wee1 and Myt1

Question 27

What activates Cdk1?

Answer 27

Aurora A kinase (AurA)/Bora activates PLK1 > activates Cdc25C > activates Cdk1

Question 28

How does ATM act in G2 to M checkpoint?

Answer 28

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)

Question 29

What co-activator complex acetylates and increases p53 in G2 to M?

Answer 29

p300/PCAF

Question 30

What does ATM phosphorylation of Chk1/2 in G2 to M do?

Answer 30

Inhibits Cdc25C > decreased Cdk1 Chk1/2 also phosphorylates p53, increasing activity, and Wee1

Question 31

How does ATR sense DNA damage?

Answer 31

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

Question 32

What is the major protein involved in late G2?

Answer 32

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

Question 33

What cell cycle checkpoint does caffeine inhibit?

Answer 33

G2 to M (more likely to proceed to mitosis)

Question 34

What is the most important cell cycle checkpoint after radiation damage?

Answer 34

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)

Question 35

What is the mitotic index?

Answer 35

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)

Question 36

What is the labeling index?

Answer 36

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)

Question 37

What technique can give the absolute duration of all cell cycle phases?

Answer 37

Percent labeled mitoses technique Flash label cells and harvest/stain at periodic intervals, plot M percentage vs time

Question 38

What is the average cell cycle time TC?

Answer 38

48 hours TS = 16 hours ~TC = 3 x TS

Question 39

What is the growth fraction?

Answer 39

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

Question 40

What is the potential doubling time?

Answer 40

Tpot = cell doubling time of a tumor assuming no cell loss Tpot = TC / GF = λ x TS / LI

Question 41

What accounts for the difference between Tpot and actual tumor doubling time (Td)?

Answer 41

Cell loss Cell loss factor φ = 1 - Tpot/Td φ = ratio of rate of cell loss vs rate of new cell production; ~77%

Question 42

How are Tpot and Td related for smaller tumors?

Answer 42

Closer together Td>Tpot for large tumors

Question 43

What is the most important factor determining tumor growth?

Answer 43

Cell loss Explains why cell cycle time is short (TC ~1-5 days) but tumor doubling time is 2-3 months

Question 44

How does the cell loss factor φ change with radiation?

Answer 44

Decreases several weeks into radiation (rate of tumor regression slows)

Question 45

Summarize tumor growth in terms of GF and cell loss.

Answer 45

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)