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

Geminin

Cdt1

HCT116cont Gem4

PhosphoChk1

PhosphoChk2

Chk1

Chk2

Loadingcontrol

Geminin

Loss of geminin leads to re-replication and activation of Chk1 and 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.

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

WHAT IS THE CELL-CYCLE?

G1 S

G2M

G0

DNA Replication

Quiescent

Mitosis

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

(S/T)PX(K/R)

P

cdk2

Cyclin

(S/T)PX(K/R)

(S/T)PX(K/R)

G1 S

G2M

G0

Cyc E Cyc D

Cyc A

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 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

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

CDK

CDK

T160

T14 Y15

CDK

CYCLIN

T160

T14 Y15

CDK

CYCLIN

T160

T14 Y15

CAK

Wee1/Mik1

CYCLINCYCLIN

CDC25 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)

CDK

CYCLIN

INACTIVE KINASE

p21/CIP1/WAF1 p27 p57

CDK4

CYCLIN D

p15 p16 p18 p19

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

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%

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

S G2 M

DNA replication

Preparation for mitosis

X

DNA replicationinterrupted

Normal

Arrestedbeforemitosis

Check-point activated by DNA damage or incomplete DNA replication inhibits

mitosis by inhibitory phosphorylation of cdk on T14 and Y15

CDK

CDK

T160

T14 Y15

CDK

CYCLIN

T160

T14 Y15

CDK

CYCLIN

T160

T14 Y15

CAK

Wee1/Mik1

CYCLINCYCLIN

CDC25 ACTIVE KINASE

S G2 M

XDNA replication interrupted

Cdc25C

ATRKinaseactivated

Chk1 kinase phosphorylated

Cdc25CPhosphatasePhosphorylated

14-3-3 bindsto phosphoCdc25Cand inhibits it

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

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

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

cdk2p21

Cyclin

K

Cy

p21 uses Cy motif to interact with cyclin-cdk2

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 18wildtype = 16

Linkers shorter than 40 A should be ineffective

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

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

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

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

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

Mevastatin induces p21 and inhibits cdk2

UUUU

Input ds RNA

siRNA (21-23 nt)

Dicer

Homologous RNAtranscripts

Degraded RNA

RNAi in flies and worms

RISC

UUUU

5’5’

Oligofectamine

21 nt RNA duplex

RNAi in mammalian cells

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)

CDK

CYCLIN

INACTIVE KINASE

p21/CIP1/WAF1 p27 p57

CDK4

CYCLIN D

p15 p16 p18 p19

INACTIVE KINASE

CDK

CDK

T160

T14 Y15

CDK

CYCLIN

T160

T14 Y15

CDK

CYCLIN

T160

T14 Y15

CAK

Wee1/Mik1

CYCLINCYCLIN

CDC25 ACTIVE KINASE

Mevastatin inhibits the activating phosphorylationof cyclin E/cdk2 on T160

…but Mevastatin does NOT inhibit the putative mammalianCAK: cyclin H/Cdk7

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?