S and M have to alternate: if not,
genomic instability
1) elevated activity of cdks
2) elevated level of geminin
S
G1
G2
3) assembly of pre-RC can
only occur in a window in
G1 (Cdc6 exported, Cdt1
degraded, Mcm2-7
phosphorylated in S)
M
4) If despite this rereplication occurs:
checkpoint pathways stop
the cell-cycle
WHAT IS THE CELL-CYCLE?
G0
Quiescent
G1
M
Mitosis
S
G2
DNA
Replication
WHY STUDY THE CELL-CYCLE IN
MEDICAL SCHOOL?
• Anomalies in the regulation of the cell-cycle are involved in
the pathogenesis of cancers
• Anomalies may be detected molecularly providing new
tools for cancer screening or detection of relapse
• Since the cell-cycle is essential for cell-proliferation,
inhibitors of the cell-cycle are anti-proliferative agents
useful in a variety of clinical settings (cancer, inflammation,
re-stenosis following angioplasty)
• Some anomalies in cell-cycle regulation predict particular
susceptibility to certain lines of therapy
The Bare Minimum
• At the heart of the cell-cycle is a dimeric enzyme which
become periodically active and inactive as the cell transits
through a given phase of the cell-cycle
• The enzyme contains a catalytic subunit called
cyclin-dependent-kinase (cdk) and a regulatory subunit
called cyclin.
Cdks phosphorylate substrates on S/T
P
(S/T)PX(K/R)
(S/T)PX(K/R)
(S/T)PX(K/R)
cdk2
Cyclin
Cyc E
Cyc D
G0
G1
S
Cyc A
M
G2
Cyc B
The Catalog
• G1 :
D1, D2 and D3 associate with cdk4 and cdk6
•
E associates with cdk2
• S:
A associates with cdk2
• M:
A and B associate with cd k1 (the old cdc2 that
started it all)
• Specialist 1: H with cdk7 is present in protein complexes
for transcription and DNA repair . Activates the other cdks
by phosphorylation
• Specialist 2: cdk5 associates with a non-cyclin protein
(p35) and is required for differentiation of neurons
• On deck: cdk8, cyclin C and G , Cdk9, cyclin T
WHAT DO THE CYCLIN-CDKS
PHOSPHORYLATE?
• Example in M: phosphorylation of nuclear lamins by cyclin
B/cdk1 results in disassembly of the nuclear lamina, a
fibrous layer that forms the wall of the nucleus
• Example in G1: phosphorylation of Rb (retinoblastoma
protein) by cyclin D/cdk4 causes it to release the
transcriptional factor E2F. The released E2F induces the
transcription of several genes essential for S phase, e.g.
ribonucleotide reductase, cyclin E etc.
Cyclin-cdks are themselves regulated by
phosphorylation of the cdk
• Cyclin associated cdk is still inactive as a kinase
• Threonine at position 160 (T160) of cdk2 has to be
phosphorylated for the kinase to be active. The cdk
activating kinase (CAK) is actually cyclin H-cdk7
• Threonine at position 14 ( T14) and tyrosine (Y15) at
position 15 of cdk2 is phosphorylated to keep the
cyclin-cdk inactive until the precise time the kinase is
required
• At that time a phosphatase, Cdc25, removes the inhibitory
phosphates and activates the cyclin-dependent kinase
T160
CDK
T14
Y15
CDK
CYCLIN
T160
CYCLIN
CAK
CDK
T14
Y15
T160
CDK
CYCLIN
T14
Y15
Wee1/
Mik1
CDC25
CYCLIN
ACTIVE KINASE
A third mode of regulation: inhibitory
proteins that associate with cyclin-cdks
• p53 (increased following DNA damage) induces the
transcription of p21/CIP1, which associates with
cyclin-cdks and inhibits the kinase activity --- another
check-point
• TGFbeta induces the transcription of p15, which associates
with cdk4 and inhibits its kinase activity
• Interferons induce the transcription of p21/CIP1
p21 family inhibits all cyclin-cdks
p16 family inhibits cyclin D-cdk4/6 (G1)
CDK4
CDK
p21/CIP1/WAF1
p27
p57
p15
p16
p18
p19
CYCLIN
CYCLIN D
INACTIVE KINASE
INACTIVE KINASE
Cancers increase activators of
cyclin-cdk
• Cyclin D is amplified or over-expressed by translocations in
parathyroid adenomas, in esophageal cancers, in breast
cancers (30-60%)
• Cyclin E is amplified or over-expressed in breast cancers
• Cdc25A is over-expressed in 30-60% of breast cancers
• Myc oncogene (8q24:14q32 translocation in Burkitt's
lymphoma; amplified in lung cancers) transcriptionally
activates the production of Cdc25A
Cancers inactivate cyclin-cdk inhibitors
• p53 (which induces p21) is inactivated by somatic
mutations in the tumors, by viral oncogenes (HPV E6)
• p53 mutation in the germ-line produces familial cancer
syndromes (e.g. Li-Fraumeni syndrome)
• p16 mutations are seen in pancreatic cancers, lung
cancers, melanomas
• Germ-line mutations in p16 lead to familial pre-disposition
to multiple tumors (MTS1), particularly melanomas.
• ATM mutations (in Ataxia-telangiectasia patients) predispose
to cancers
Small
Esoph- Gli- Head
cell
ageal oma &
Ca lung Ca
Neck
p16 loss 10%
p16:cdk4
Cyclin D
Cyclin D:cdk4
Rb-P
Rb:E2F
E2F
30%
cdk4
amplified
Cyclin D 5%
amplified
Rb
85%
loss
Activates transcription
55% 20%
20%
35%
45%
Diagnosis/Prognosis
• Use in screening: PCR based detection of populations with
anomalies in cell-cycle regulators e.g. L.O.H. of p16, cyclin
over-expression, amplification of a gene
• Detection of relapse/minimal residual disease
• Use in prognosis: e.g. tumors with high S phase fraction
detected by flow cytometry have poorer prognosis
• Use in predicting responsiveness to a particular type of
therapy: e.g. high S phase fraction and loss of p53 will
make cells more suceptible to DNA damaging agents
Therapy
• Pharmaceutical companies are screening for chemicals
that inhibit cdk2 kinase and CDC25 phosphatase.
Potential new chemotherapeutic agents
• Adenovirus engineered to have no E1b gene will only grow
in cells without p53. Thus specifically infect and destroy
tumor cells
• Crystal structure of p21 with cyclin-cdk solved. The way
p21 binds to the kinase may be copied by designer
chemicals which will be cdk inhibitors
DNA replication Checkpoint
G2
S
M
DNA replication
Preparation for mitosis
X
DNA replication
interrupted
Arrested
before
mitosis
Normal
Check-point activated by DNA damage
or incomplete DNA replication inhibits
mitosis by inhibitory phosphorylation of
cdk on T14 and Y15
T160
CDK
T14
Y15
CDK
CYCLIN
T160
CYCLIN
CAK
CDK
T14
Y15
T160
CDK
CYCLIN
T14
Y15
Wee1/
Mik1
CDC25
CYCLIN
ACTIVE KINASE
Cdc25C
ATR
Kinase
activated
X
DNA replication
interrupted
M
G2
S
Cdc25C
Phosphatase
Phosphorylated
Chk1
kinase
phosphorylated
14-3-3s binds
to phosphoCdc25C
and inhibits it
Cdt1
Geminin
Loss of geminin leads to re-replication and activation of Chk1 and Chk2
HCT116
cont Gem4
Loading
control
Geminin
Phospho
Chk1
Chk1
Phospho
Chk2
Chk2
Depletion of geminin activates G2/M checkpoint,
resulting in sequestration of Cdc25C outside the
nucleus (red on right panel: cytoplasmic Cdc25C).
Rereplication by depletion of geminin activates
the G2/M checkpoint.
Regulated proteolysis is an important component of cell-cycle regulation
K
K
Ubiquitinylation
by an E3
ubiquitin ligase:
SCF in G1 and S
APC in M
Proteasome
recognizes
polyubiquitinylated
substrate and
degrades it
Examples of substrates degraded in this manner:
G1: Cdk inhibitor, p27
S: Cdt1
M : securin, a molecule that inhibits the protease that separates daughter
chromosomes
cyclin A, cyclin B
Cell Cycle Checkpoints
G1
S
G2
Metaphase
Anaphase
CDK1
APC
Chromosome
condensation
Sister chromatid
separation
CDK2
p53  p21
Repair of DNA damage
G1 DNA damage
checkpoint
ATRChk1
ATMChk2
DNA replication;
Repair of damage
DNA replication
checkpoint
Kinetochore
attachment
S DNA damage
checkpoint
Spindle
checkpoint
Therapy
• Pharmaceutical companies are screening for chemicals
that inhibit cdk2 kinase and CDC25 phosphatase.
Potential new chemotherapeutic agents
• Adenovirus engineered to have no E1b gene will only grow
in cells without p53. Thus specifically infect and destroy
tumor cells
• Crystal structure of p21 with cyclin-cdk solved. The way
p21 binds to the kinase may be copied by designer
chemicals which will be cdk inhibitors
p21 uses Cy motif to interact with cyclin-cdk2
K
p21
cdk2
Cy
Cyclin
Chen, MCB 2002
Crystal structure of cdk inhibitor p27N
in complex with cyclin A/Cdk2
Pavletich. Nature 1996
Effect of Linker Length on Substrate Phosphorylation
40 A
- (X)n
n = 2, 6, 12, or 18
wildtype = 16
Linkers shorter than 40 A should be ineffective
Cy peptides inhibit Cyclin-Cdk2
Chen et al. 1996, MCB
Cy peptide Competitively Inhibits Cyclin E/cdk2 and
Cyclin A/cdk2
Cyclin E/cdk2
12
Cyclin A/cdk2
8
Ki = 7.5 ± 0.5 µM
1/v0 (pmol/min)-1
10
1/v0 (pmol/min)-1
Ki = 117.5 ± 11.6 µM
7
8
6
4
6
5
4
3
2
2
1
0
-0.2
-0.1
0
0
0.1
0.2
1/[CDC6(wt)] (µM)-1
0.3
0.4
-0.2
-0.1
0
0.1
0.2
1/[CDC6(wt)] (µM)-1
0.3
0.4
A new class of cdk inhibitors
Existing cdk inhibitors are all ATP mimetic chemicals
that competitively inhibit the binding of ATP to the cdk2
Cy mimetic chemicals will be a new class of cdk
inhibitors :
•specific for sub-classes of substrates
•specific for a given cyclin that might be de-regulated
in a cancer
•could synergise with ATP mimetic chemicals.
Effect of statins on prostate cancer cells
Statins are widely used (FDA approved!) chemicals that inhibit
HMG CoA reductase and reduce the levels of cholesterol:
Fluvastatin (Lescol) - Novartis
Atorvastatin (Lipitor) - Pfizer
Simvastatin (Zocor) - Merck
Pravastatin (Pravachol) - Bristol Myers Squibb
Lovastatin (Mevacor) - Merck
They also have anti-proliferative effect on epithelial cells
Mevastatin blocks prostate cancer cell PC3 at G1-S
90
80
70
60
50
G1
S
G2/M
40
30
20
10
0
Control/24h
Control/36h
Mev/24h
Mev/36h
Mevastatin induces p21 and inhibits cdk2
RNAi in flies and worms
Input ds RNA
Dicer
UU
UU
siRNA (21-23 nt)
RISC
Homologous RNA
transcripts
Degraded RNA
RNAi in mammalian cells
5’
UU
Oligofectamine
21 nt RNA duplex
UU
5’
RNAi of p21 prevents the induction of p21 by mevastatin
RNAi of p21 does not prevent the G1-S block and
Rb dephosphorylation induced by mevastatin
p21 family inhibits all cyclin-cdks
p16 family inhibits cyclin D-cdk4/6 (G1)
CDK4
CDK
p21/CIP1/WAF1
p27
p57
p15
p16
p18
p19
CYCLIN
CYCLIN D
INACTIVE KINASE
INACTIVE KINASE
T160
CDK
T14
Y15
CDK
CYCLIN
T160
CYCLIN
CAK
CDK
T14
Y15
T160
CDK
CYCLIN
T14
Y15
Wee1/
Mik1
CDC25
CYCLIN
ACTIVE KINASE
Mevastatin inhibits the activating phosphorylation
of cyclin E/cdk2 on T160
…but Mevastatin does NOT inhibit the putative mammalian
CAK: cyclin H/Cdk7
Summary of the mechanism by which statins inhibit the
cell-cycle in prostate cancer cells
•Mevastatin blocks the cell-cycle at G1-S transition
•Rb is de-phosphorylated, cyclin D1/cdk4 unaffected, cyclin E/cdk2
inhibited and cyclin A downregulated
•p21 is induced, but not necessary for cyclin E/cdk2 inhibition
•T160 phosphorylation is inhibited, but the conventional CAK cyclin
H/cdk7 is active
•T160-P phosphatase activity is not increased
•Do statins affect a new (undiscovered) CAK?
Unsolicited advice to future scientists
1) Always remember: you are in charge
2) You are not doing this for money or for nice lifestyle
3) Scientific research is the most exciting enterprise that the
human race has devised: you are part of a grand tradition
4) Be an optimist: no one else will be
5) There is no substitute for work at the bench AND for keeping
on top of the Literature