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Transcript of CDK1 Chromosome condensation Sister chromatid separation CDK2 DNA replication; Repair of damage APC...
![Page 1: CDK1 Chromosome condensation Sister chromatid separation CDK2 DNA replication; Repair of damage APC DNA replication checkpoint Spindle checkpoint SG2MetaphaseAnaphase.](https://reader030.fdocuments.net/reader030/viewer/2022033104/5697bf871a28abf838c88b7b/html5/thumbnails/1.jpg)
CDK1
Chromosomecondensation
Sister chromatidseparation
CDK2
DNA replication;Repair of damage
APC
DNA replicationcheckpoint
Spindlecheckpoint
S G2 Metaphase Anaphase
Kinetochoreattachment
Repair of DNA damage
G1
G1 DNA damagecheckpoint
Cell Cycle Checkpoints
p53 p21
S DNA damagecheckpoint
ATRChk1ATMChk2
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Geminin
Cdt1
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HCT116cont Gem4
PhosphoChk1
PhosphoChk2
Chk1
Chk2
Loadingcontrol
Geminin
Loss of geminin leads to re-replication and activation of Chk1 and Chk2
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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.
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S and M have to alternate: if not, genomic instability
S
M
G1 G2
1) elevated activity of cdks
2) elevated level of geminin
3) assembly of pre-RC can only occur in a window in G1 (Cdc6 exported, Cdt1 degraded, Mcm2-7 phosphorylated in S)
4) If despite this re-replication occurs: checkpoint pathways stop the cell-cycle
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WHAT IS THE CELL-CYCLE?
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G1 S
G2M
G0
DNA Replication
Quiescent
Mitosis
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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
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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.
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Cdks phosphorylate substrates on S/T
(S/T)PX(K/R)
P
cdk2
Cyclin
(S/T)PX(K/R)
(S/T)PX(K/R)
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G1 S
G2M
G0
Cyc E Cyc D
Cyc A
Cyc B
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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 cdk1 (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
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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.
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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
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CDK
CDK
T160
T14 Y15
CDK
CYCLIN
T160
T14 Y15
CDK
CYCLIN
T160
T14 Y15
CAK
Wee1/Mik1
CYCLINCYCLIN
CDC25 ACTIVE KINASE
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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
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p21 family inhibits all cyclin-cdks p16 family inhibits cyclin D-cdk4/6 (G1)
CDK
CYCLIN
INACTIVE KINASE
p21/CIP1/WAF1 p27 p57
CDK4
CYCLIN D
p15 p16 p18 p19
INACTIVE KINASE
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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
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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
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Cyclin D
Rb:E2F
Rb-P E2F
Cyclin D:cdk4
p16:cdk4
20%
p16 loss
cdk4 amplified
Cyclin D amplified
Rb loss
Small cell Ca lung
Esoph- ageal Ca
10%
5%
85%
30%
35%
Gli- oma
55%
Activates transcription
Head & Neck
20%
45%
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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
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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
![Page 23: CDK1 Chromosome condensation Sister chromatid separation CDK2 DNA replication; Repair of damage APC DNA replication checkpoint Spindle checkpoint SG2MetaphaseAnaphase.](https://reader030.fdocuments.net/reader030/viewer/2022033104/5697bf871a28abf838c88b7b/html5/thumbnails/23.jpg)
DNA replication Checkpoint
S G2 M
DNA replication
Preparation for mitosis
X
DNA replicationinterrupted
Normal
Arrestedbeforemitosis
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Check-point activated by DNA damage or incomplete DNA replication inhibits
mitosis by inhibitory phosphorylation of cdk on T14 and Y15
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CDK
CDK
T160
T14 Y15
CDK
CYCLIN
T160
T14 Y15
CDK
CYCLIN
T160
T14 Y15
CAK
Wee1/Mik1
CYCLINCYCLIN
CDC25 ACTIVE KINASE
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S G2 M
XDNA replication interrupted
Cdc25C
ATRKinaseactivated
Chk1 kinase phosphorylated
Cdc25CPhosphatasePhosphorylated
14-3-3 bindsto phosphoCdc25Cand inhibits it
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Ubiquitinylation by an E3 ubiquitin ligase:
SCF in G1 and S
APC in M
Proteasome recognizes polyubiquitinylated substrate and degrades it
K K
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
Regulated proteolysis is an important component of cell-cycle regulation
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CDK1
Chromosomecondensation
Sister chromatidseparation
CDK2
DNA replication;Repair of damage
APC
DNA replicationcheckpoint
Spindlecheckpoint
S G2 Metaphase Anaphase
Kinetochoreattachment
Repair of DNA damage
G1
G1 DNA damagecheckpoint
Cell Cycle Checkpoints
p53 p21
S DNA damagecheckpoint
ATRChk1ATMChk2
![Page 29: CDK1 Chromosome condensation Sister chromatid separation CDK2 DNA replication; Repair of damage APC DNA replication checkpoint Spindle checkpoint SG2MetaphaseAnaphase.](https://reader030.fdocuments.net/reader030/viewer/2022033104/5697bf871a28abf838c88b7b/html5/thumbnails/29.jpg)
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
![Page 30: CDK1 Chromosome condensation Sister chromatid separation CDK2 DNA replication; Repair of damage APC DNA replication checkpoint Spindle checkpoint SG2MetaphaseAnaphase.](https://reader030.fdocuments.net/reader030/viewer/2022033104/5697bf871a28abf838c88b7b/html5/thumbnails/30.jpg)
cdk2p21
Cyclin
K
Cy
p21 uses Cy motif to interact with cyclin-cdk2
Chen, MCB 2002
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Crystal structure of cdk inhibitor p27N in complex with cyclin A/Cdk2
Pavletich. Nature 1996
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Effect of Linker Length on Substrate Phosphorylation
40 A
- (X)n -
n = 2, 6, 12, or 18wildtype = 16
Linkers shorter than 40 A should be ineffective
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0
25
50
75
100
Peptide (nM)
A/K2; PS103
A/K2; PS102
A/K2; PS101
A/K2; PS100
E/K2; PS101
E/K2; PS100
Cy peptides inhibit Cyclin-Cdk2
Chen et al. 1996, MCB
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0
2
4
6
8
10
12
-0.2 -0.1 0 0.1 0.2 0.3 0.4
1/v 0
(p
mo
l/m
in)-1
1/[CDC6(wt)] (µM)-1
0
1
2
3
4
5
6
7
8
-0.2 -0.1 0 0.1 0.2 0.3 0.41/[CDC6(wt)] (µM)-1
1/v0
(p
mo
l/m
in)-
1
Cy peptide Competitively Inhibits Cyclin E/cdk2 and Cyclin A/cdk2
Ki = 7.5 ± 0.5 µM Ki = 117.5 ± 11.6 µM
Cyclin E/cdk2 Cyclin A/cdk2
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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.
A new class of cdk inhibitors
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Statins are widely used (FDA approved!) chemicals that inhibit HMG CoA reductase and reduce the levels of cholesterol:
Fluvastatin (Lescol) - NovartisAtorvastatin (Lipitor) - PfizerSimvastatin (Zocor) - MerckPravastatin (Pravachol) - Bristol Myers SquibbLovastatin (Mevacor) - Merck
They also have anti-proliferative effect on epithelial cells
Effect of statins on prostate cancer cells
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0
10
20
30
40
50
60
70
80
90
Control/24h Control/36h Mev/24h Mev/36h
G1SG2/M
Mevastatin blocks prostate cancer cell PC3 at G1-S
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Mevastatin induces p21 and inhibits cdk2
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UUUU
Input ds RNA
siRNA (21-23 nt)
Dicer
Homologous RNAtranscripts
Degraded RNA
RNAi in flies and worms
RISC
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UUUU
5’5’
Oligofectamine
21 nt RNA duplex
RNAi in mammalian cells
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RNAi of p21 prevents the induction of p21 by mevastatin
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RNAi of p21 does not prevent the G1-S block and Rb dephosphorylation induced by mevastatin
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p21 family inhibits all cyclin-cdks p16 family inhibits cyclin D-cdk4/6 (G1)
CDK
CYCLIN
INACTIVE KINASE
p21/CIP1/WAF1 p27 p57
CDK4
CYCLIN D
p15 p16 p18 p19
INACTIVE KINASE
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CDK
CDK
T160
T14 Y15
CDK
CYCLIN
T160
T14 Y15
CDK
CYCLIN
T160
T14 Y15
CAK
Wee1/Mik1
CYCLINCYCLIN
CDC25 ACTIVE KINASE
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Mevastatin inhibits the activating phosphorylationof cyclin E/cdk2 on T160
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…but Mevastatin does NOT inhibit the putative mammalianCAK: cyclin H/Cdk7
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Summary of the mechanism by which statins inhibit thecell-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?