Cell Cycle – Control of Cell Proliferation...1 fertilized egg ! ! !!100 000 000 000 000 cells Cell...

Cell Cycle – Control of Cell Proliferation Ludger Hengst

Sektion für Medizinische Biochemie - Biozentrum - Medizinische Universität Innsbruck

1 fertilized egg !! !! ! !100 000 000 000 000 cells �

Cell cycle – control of cell proliferation Ludger Hengst

Biozentrum - Medizinische Biochemie - Medizinische Universität Innsbruck

In Adults: ! ≈4.000.000 cell divisions / sec.�

1.   Principles of the cell cycle �

Cell Cycle Checkpoints�

2.  The restriction point�

3.   CDKs – central cell cycle regulators�

4.   CDK inhibitors�

5.   The retinoblastoma protein�

6.   The RB-E2F pathway�

1.  Duplication of the genome!

2.  Separation of the duplicated genetic material (and other cellular compounds) into two daughter cells"

Central Aims of the Cell Cycle

Four Cell Cycle Phases �

M

Mitosis and Cytokinesis"

S

DNA Synthesis

G1

G2

Gap-phase 1"

Gap-phase 2"

M

S

G1

G2

Principles of the eucaryotic cell cycle:

1.  One and only one replication of the entire genome per cell cycle

2.  Initiation of a subsequent cell cycle phase requires proper termination of the preceding phase

M

S

G1

G2 G0

Restriction point"

G0-phase

Why seperate cell cycle phases?

The temporal separation of DNA replikation and mitosis permits the incorporation of control mechanisms in the eucaryotic cell cycle. These control mechanisms are called!Checkpoints.

Checkpoint: a process within the cell cycle, which controls the transition ! from one cell cycle state into the next state

!! - Checkpoints secure e.g. that DNA replication is completed ! before mitosis can be initiated.

!! - Checkpoints secure genomic stability by arresting cell ! cycle progression upon DNA damage.!

Checkpoints

G1/S Checkpoint"

√ size ? "

√ DNA damage ?"

G2 / M Checkpoint"√ DNA replicated?"

√ DNA damage?"

Spindel Checkpoint"√ all chromosomes attached to the spindle ?"

G1

G0

S

M

G2

Important Checkpoints

√ mitogens anti-mitogenic signals ?""√ differentiation - signals ?"

1.  Restrict cell divisions to precisely the required numbers, e.g. induced divisions after wounding or cell death�"- Avoids hypo- and hyperproliferation�

�2.  Warrant one, and only one DNA replication of all regions of

the genome per cell cycle. �"- Avoids genetic instability�

�3.  Precise segregation of the genome into both daughter cells.�

"- Avoids genetic instability��4. "Prevention of replication or segregation of damaged DNA �

"- Avoids the multiplication of damaged DNA

Important aims of cell cycle control mechanisms: �

Consequences of misregulated cell cycle control: �Genetic instability of tumor cells

Aneuploidy : abnormal number of chromosomes (extra or missing)

Chromosomal translocations: Rearrangements of chromosome parts between nonhomologues chromosomes

1

3

14

Length of cell cycle phases"

Duplication of bacteria:"

Eucaryotes:"Early frog embryo cells " 30 minYeast " " " 1.5 - 3 hCells in the epithelium of the small intestine " 12 h"Proliferating mammalian cells18-24 h"Fibroblasts in culture " 20 h"Human liver cells " " 1 year"

E. coli: 20 - 25 min"

M

G1"

G2"

S

1 h"

6-12 h"

6-8 h"

2-4 h"

G1

G0

S

M

G2

Restriction Point"

The Cell Cycle �

Mitosis and cytokinesis"

DNA-Synthesis

Mitogens ?"Antimitogens ?"

Growth factors ?"Differentiation signals ?"

Exit from the cell cycle:"- senescence - terminal differentiation- quiescence""G0-phase / quiescence:"-  no cell divisions"-  no DNA-replikation"- can persist hours, days or years"

Cell Fusions

Rao, P.N. & Johnson, R.T. (1970). Mammalian Cell Fusion: Studies on the Regulation of DNA Synthesis and Mitosis. Nature 225, 159-164. Johnson, R.T. & Rao, P.N. (1970). Mammalian Cell Fusion: Induction of Premature Chromosome Condensation in Interphase Nuclei. Nature 226, 717-722.

Fusion of G1, S or G2 cells with mitotic cells leads to nuclear envelope breakdown and DNA condensation in the heterocaryon.!!Mitotic cells contain a factor (“MPF” - Mitosis promoting factor) which can induce mitosis in cells of other cell cycle phases.!

Rao, P.N. & Johnson, R.T. (1970). Mammalian cell fusion: studies on the regulation of DNA synthesis and mitosis.Nature 225, 159-164. Johnson, R.T. & Rao, P.N. (1970). Mammalian cell fusion: induction of premature chromosome condensation in interphase nuclei.Nature 226,

717-722.

Heterocaryon experiments"

G1, S or G2

+ M M

Fusion of G1 cells with S-phase cells results in heterocaryons, in which the G1 nuclei initiate DNA replikation. S-phase cells only initiate mitosis after completion of DNA!replication in G1 cells.!!S-phase cells contain a factor which can initiate DNA replication in G1 cells. Checkpoints prevent “premature” entry into mitosis before S-phase is completed in both nuclei.


717-722.


G1, S or G2

+ M

G1

+

S S

M


717-722.


G1, S or G2

+ M

G1

+

S S

S

+

G2 no DNA synthesis in the G2 nucleus, delayed entry into mitosis

M

G2 nuclei do not re-initiate DNA replication!Existence of a “licensing system”, which labels replicated DNA

and prevents re-replication.

The Nobel Prize in Physiology or Medicine 2001 "for their discoveries of key regulators of the cell cycle"

R. Tim Hunt

Fission yeast

Sir Paul M. Nurse

Sea Urchin

Leland H. Hartwell

Bakers yeast

The Cell Cycle Engine�

Cyclin A (N-term.)

Cdk2

Cdk

Cyclin

The Cell Cycle Engine: Cyclin-Dependent Kinases�

time!

rel. Cdk-kinase activity

Cell Cycle Transitions are driven by the oscillating Activity� of Cyclin-Dependent Kinases (CDKs) �

G1-phase kinase! S-phase kinase! M-phase kinase!

G 1 S G 2 M

G1

G0

S

M

G2

Cdk2 Cyc E

Cdk2 Cyc A

Cdk1 Cyc A

Cdk1 Cyc B

Cdk4 Cyc D

Restriction Point"

The Cell Cycle Engine: Cyclin-Dependent Kinases�

inactive! inactive! inactive!active!

Assembly factors ?spontanious"

CAK"KAP!

Wee1 / Myt1"Cdc25!

Cdk

Cyclin

Cdk

Cyclin

Cdk

Cyclin

Cdk

Cyclin

P"P"P"P"

Kinases"Phosphatases!

CDKs are regulated by activating and inhibitory phosphorylations �

M

G2

S

G1

G1 arrest"(Lovastatin; TGF-β)"

Molecular Brakes: CDK Inhibitor Proteins �

G1

G2 S

M

100!

75!

50!

25!

0!

% E-

kina

se a

ctiv

ity!

Cdk

Cyclin p27

Hengst et al., PNAS 1994!

Mechanism of CDK inhibition by p27 �

Cyclin A

Cdk2

p27

Cyclin A Cdk2

p27Kip1 Cdk

Cyclin p27

Mechanism of CDK inhibition by p27 �

ATP"p27"

Lacy et al., Nature Structural & Molecular Biology. 2004

Cip/Kip Inhibitors are Intrinsically Unstructured Proteins (IUPs)!

p27 198 AA

CDK inhibitory domain

p21 p27 p57

22 30 40 50 60 70 80 90 PCGSK ACR R LFGPVD S E Q L S R D SDALM A GCIQ E ARE R W N F D F VTET PL E-- G DFA W ER V RGLG L P KL Y LPTGP EHPKP S ACR N LFGPVD H E E L T R DL EKHC RDMEE AS Q RK W N F D F Q NHK PL E-- G KYE W Q E V EKG SL P EF Y YRPPR VLVRT S ACR S LFGPVD H E E L G R E L RMRA A ELNA E D Q N R W D F N F Q QDV PL RGP G RLQ W M E V DSE S V P AF Y RET-V

p57

Cyclin binding domain CDK binding domain

316 AA

p21 164 AA

The Cip/Kip-Family of CDK Inhibitors

Cdk

Cyclin p27

Linker helix

Two Families of CDK Inhibitors �

Cdk p16

Ink4 family!(Inhibitor of Cdk4)!

p15, p16, p18, p19 (Ink4 a-d)!!-  specific for cyclin D / CDK4,6!-  bind the CDK subunit!-  ankyrin repeat structure!!

Cdk

Cyclin p27

Cip/Kip family (Cdk interacting protein;!Kinase inhibitory protein)!

p21, p27, p57 (Cip1, Kip1, Kip2)!!-  bind to a broad spectrum of CDK/ cyclins!-  bind the CDK / cyclin complex!-  conserved N-terminal CDK- inhibitory domain!-  may act as actvators for cyclin D/Cdk4,6 !

Cdk6

p16Ink4a

INK4 CDK4,6 Inhibitors�

Cdk4 p16

Cyclin D

What are critical CDK substrates at the G1/S transition ? �

Rb

The CDK substrat retinoblastom protein (Rb) is a central cell cycle regulator

E1A G1

G0

S

M

G2

-  Retinoblastoma is a rapidly developing childhood cancer (1/20.000) arising from immature retinal cells -  The Rb protein is a tumor suppressor protein; 45% of the patients carry a heterozygous mutation in Rb1 -  Children with (heteroygous) mutations in Rb frequently develop tumours in other tissues (Knudsons two-hit hypothesis) -  Viral oncoproteins like E1A and HPV E7 bind Rb and induce cell division in cultured cells

Rb binds E2F and inhibits cell cycle progression

Rb E2F

E2F binding site

inactive

Rb

E2F binding site

E2F

active

E1A

Phosphorylation of Rb releases E2F

inactive

P

CDK

Rb E2F

E2F binding site

P Rb

E2F binding site

E2F

active

Rb E2F

mitogens

P

Mitogen induced expression and activation of cyclin D/ CDK initiates cell cycle progression

G1

G0

S

M

G2

Cdk2 Cyc E

Cdk2 Cyc A

Cdk1 Cyc A

Cdk4 Cyc D

P

Cdk4 Cyc D

Cdk2 Cyc E

mitogen independent

CDK inhibitors prevent CDK activation

Rb E2F

G1

G0

S

M

G2

Cdk2 Cyc E

Cdk4 Cyc D

p16 p15

p18 p19

p27 p57

p21

Cdk4

STOP

The Rb pathway during the G0/G1/S transition"

S-Phase Cell

S G0/G1

p27

Antimitogens

Cdk4,6 p16

Antimitogens

E2Fs Rb

HDAC

Cdk4,6

Cdc37

p27 degradation

P

S-Phase Cell

S

Cdk2 Cyc E

Cdk4,6 p27

Cyc D

Cdk2 Cyc E

p27

HDAC

E2Fs Rb P

P P

Cyclin E DHFR DNA Pol.α

G1

Cyclin A Cdk1

E2Fs

P

P Rb P

P P

P P

Cyc D

Mitogens

Cdk4,6 p16

Antimitogens

Antimitogens

Der Rb Signalweg des G0/G1/S Übergangs"

Head and neck!

Prostate!

Lung! Melanoma!

Gastrointestinal!

Breast!

Liver!Pancreas!

Bladder! Bone (osteosarcoma)!

Bone marrow (leukemia)!

Lymphoma!

p130"

p130" p130"

p16"

p16"

p16"

p15"

p15"

p15"

D1"

Pituitary!

Testis/Ovary!

p16"

D2"

Cdk6"

Cdk6"

D1"

D1"

D1"

p16"

p16"

p130"

D1"

Cdk4"p16"

p16"

D1"D3"

p16"

Cdk4"

p16"D1"

Cdk4" D1"

D1"

Cdk4" p16"

D1"

Cdk4" D1" p16"

pRb"E1"

p27"

p27"

p27"

p27" E1"

E1"

E1"p27"

E1"p27"

p27"

E1" p27"

E1" p27"

Cdk4"Cdk2"D2"

Other Sarcomas!p16"

Endometrium!p130"Cdk4"

p27"D1"

E1"

Glioma/blastoma!Cdk4"Cdk6"E1" p16"p15"

70%

> 80%

> 90%

> 80% > 90%

> 90%

> 80%

> 90%

> 90%

D2" D3" E1"> 90%

20%

> 80%

> 80%

p16" > 80%

E1"70%

Cdk4" E1"90%

> 90%

pRb"

pRb"

pRb"

pRb"

pRb"

pRb"

pRb"

pRb"

pRb"

pRb"

p27"

p27"

D1"

p27"

E1"

p16"

p27"

p27"

*

*

*

* *

*

*

*

*

*

*

* *

*

*

Cdk2"

*

Cdk4"p15"

* * * * *

* * D1"

* *

E1"

* *

* *

D3"

*

* *

*

pRb"D1"p16"

* * *

Cancer is a disease of the cell cycle �

From: Malumbres and Barbarcid: Nature Reviews Cancer (01). Vol1, 222

Questions ��

???"

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