CST Guide:

Section I: Research Areas
chapter 03: Cell Growth and Death
Cell Cycle, Checkpoint
Control, and DNA Damage
Control of eukaryotic cell growth and division involves molecular circuits known as “checkpoints” that
ensure proper timing of cellular events. Passage through a checkpoint from one cell cycle phase to the
next requires a coordinated set of proteins that monitor cell growth and DNA integrity. Uncontrolled cell
division or propagation of damaged DNA can contribute to genomic instability and tumorigenesis.
Phases of the Cell Cycle
G2/M Checkpoint
The G2/M checkpoint prevents cells containing damaged DNA from entering mitosis (M). Activated
CDK1 (cdc2) bound to cyclin B promotes entry into M-phase. Wee1 and Myt1 kinases and cdc25
phosphatase competitively regulate CDK1 activity; Wee1 and Myt1 inhibit CDK1 and prevent entry into
M-phase, while cdc25 removes inhibitory phosphates. DNA damage activates multiple kinases such
as ATM/ATR, DNA-PK, HIPK2 that phosphorylate kinases Chk1/2 and tumor suppressor protein p53.
Chk1/2 kinases stimulate Wee1 activity and inhibit cdc25C, preventing entry into M-phase. Phosphorylation
of p53 promotes dissociation between p53 and MDM2 and allows binding of the transcription
factor to DNA.
UV treatment results
in phosphorylation of
ATM at Ser1981.
kDa
200
Phospho-ATM
(Ser1981)
G2
Preparation
for Mitosis
Prophase
Metaphase
Anaphase
M
Cyclin B1/CDK1 complex is expressed in replicating Jurkat cells.
Cyclin B1 (D5C10) XP ® Rabbit mAb #12231: Flow cytometric analysis of
Jurkat cells using #12231 and Propidium Iodide (PI)/RNase Staining Solution
#4087 (DNA content). Anti-rabbit IgG (H+L), F(ab’) 2
Fragment (Alexa Fluor ®
488 Conjugate) #4412 was used as a secondary antibody.
Cyclin B1
10 4
10 3
10 2
140
100
80
200
ATM
Telophase
10 1
140
G 0
10 0 0 200 400 600 800 1000
DNA (PI)
100
S
DNA
Replication
Preparation
for DNA
Synthesis
G1
UV treatment results in nuclear translocation of Phospho-p53.
Phospho-p53 (Ser15) (16G8) Mouse
mAb (Alexa Fluor ® 555 Conjugate)
#9481: Confocal IF analysis of HT-29
cells, untreated (left) or UV-treated
(right), using #9481 (red). Actin filaments
were labeled with Alexa Fluor ®
488 Phalloidin #8878 (green).
80
– + UV
Phospho-ATM (Ser1981) (D6H9)
Rabbit mAb #5883: WB analysis of
extracts from 293 cells, untreated or
UV-treated (100 mJ, 4 hr recovery),
using #5883 (upper) or ATM (D2E2)
Rabbit mAb #2873 (lower).
CDK inhibitor p27
Kip1 is expressed
in quiescent cells
and degraded in
mitotic cells.
p27 Kip1 (D69C12) XP ® Rabbit
mAb #3686: Flow cytometric
analysis of Jurkat cells using
#3686 versus Propidium Iodide
(PI)/RNase Staining Solution
#4087. Anti-rabbit IgG (H+L),
F(ab’) 2 Fragment (Alexa Fluor ®
488 Conjugate) #4412 was
used as a secondary antibody.
G1/S Checkpoint
The G1/S checkpoint controls progression of cells through the restriction point (R) into the DNA
synthesis S-phase. During G1, the tumor suppressor Rb binds and inhibits transcription factor E2F.
Phosphorylation of Rb by cyclin-bound cyclin dependent kinases (CDK) in late G1 induces dissociation
of Rb and permits E2F-mediated transcription of S-phase-promoting genes. Responding to upstream
signals, INK4 and Kip/Cip family inhibitors control CDK activity and prevent entry into S-phase. DNA
damage activates response pathways through ATM/ATR and Chk1/2 kinases to block CDK activity,
leading to cell cycle arrest and DNA repair or cell death.
p27 Kip1
10 4
10 3
10 2
10 1
10 0 0 200 400 600 800 1000
DNA (PI)
Serum treatment results in
phosphorylation of Rb at Ser807/811.
Phospho-Rb (Ser807/811)
(D20B12) XP ® Rabbit mAb
#8516: WB analysis of extracts
from WI-38 cells using #8516.
Lanes
1. Serum-starved for 3 days (-)
2. Serum-starved for 3 days
followed by 10% serum for
2 days (+)
kDa
200
140
100
80
60
1 2
Phospho-Rb
(Ser807/811)
Spindle Checkpoint
As mitosis proceeds, mitotic spindles are assembled that connect the kinetochore regions of sister
chromatids to the microtubules within the spindle, centering the chromatids along the metaphase
plate. Many proteins comprise the kinetochore and play a role in this process, including the histone H3
variant CENP-A, survivin, and Aurora B kinase, which is itself regulated by the microtubule nucleating
protein TPX2. The spindle checkpoint ensures proper chromatid attachment prior to progression from
metaphase to anaphase. The SCF and APC/C protein complexes play prominent roles in the spindle
checkpoint, with APC-cdc20 initiating the entry into anaphase by promoting ubiquitin-mediated degradation
of multiple substrates, including cyclin B and the regulatory protein securin.
Chemical Modulators for Studies of the Cell Cycle and DNA Damage
#9886 Docetaxel Inhibits microtubule depolymerization; prevents cell division
#5927 Doxorubicin Inhibits DNA and RNA synthesis by intercalating the DNA helix; inhibits topoisomerase I
#2200 Etoposide Inhibits topoisomerase II resulting in DNA breakage; induces apoptosis
#2194 MG-132 Potent inhibitor of the proteasome and calpain
#2190 Nocodazole Inhibits microtubule polymerization; induces cell cycle arrest in G2/M-phase
#9807 Paclitaxel Inhibits microtubule depolymerization; prevents cell division
#9885 Roscovitine Potent and selective inhibitor of CDK1, 2, and 5 (ATP-competitive)
TPX2 regulates
activity of Aurora B,
a kinase critical for
proper chromatid
attachment to the
mitotic spindle.
TPX2 (D2R5C) XP ® Rabbit mAb
#12245: Confocal IF analysis of MCF7
cells using #12245 (green). Actin filaments
were labeled with DY-554 phalloidin
(red). Blue pseudocolor = DRAQ5 ®
#4084 (fluorescent DNA dye).
98 For Research Use Only. Not For Use in Diagnostic Procedures. See pages 302 & 303 for Pathway Diagrams, Application, and Reactivity keys.
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