1

1

Cell CycleRegulation and Checkpoints

Chi-Wu Chiang, Ph.D.IMM, NCKU

基醫所

2

2

-----Accompanied with cell division, cell growth and cell death

Cell mass=cell number+cell size

cell mass

From cell division to a full grown body

3

3

Cell division, cell growth, and cell death areindependently regulated but linked

Intrinsic programs

Extracellular signal molecules

Regulated cell cycle progression

Programmed cell death

Mitogens

Growth factors

Survival factors

Apoptosis-inducing factorsProliferation inhibitors

4

4

Introduction to cell cycle and cell cyclecheckpoints

5

5

Cell cycle

A cell reproduces by performing an orderly sequence of events in whichit duplicates its contents and then divides in two

6

6

The phases of cell cycle

7

7

M

S

M

G2 G1

S

Early embryonic cell cycle

Somatic cell cycle

Embryonic cell cycle and somatic cell cycle

12h

6h

6h

30 min

8

8

Yeast provides a good genetic system for studyon cell cycle

Mostly proliferating in a haploid state

Easy for genetic manipulation

Cdc genes and cdc mutants ----cell-division-cycle genes

9

9

Cdc mutants selected by temperaturesensitive mutations

Temperature-sensitive mutantsFunctioning in permissive condition (low temp.) andnonfunctioning in restrictive condition (high temp.)

A mutant that cannot complete the cell cycle, cannot bepropagated

10

10

Xenopus oocyte: a giant cell for study cell cyclebiochemically

1 mm in diameterCarrying 100,000 times more cytoplasm than an average cell in thehuman body

After fertilization occurs, the first division takes about 90 minutes,and the next 11 divisions occur at 30-mim intervals, producingabout 4096 cells within 7 hours

Each cycle is divided into S and M phase without detectable G1 or G2phases

11

11

Studying the cell cycle in a cell-free system

Observation of repeated nuclei decondense and DNA replication and mitosisIn vitro

12

12

Measurements of cell cycle progression

Propidium iodide (PI) staining

EMBO 2003, 22:5459

Oncogene 2001, 20:4507

BrdUstaining(bromo-deoxyuridine)

PIstaining

13

13

The cell-cycle control system

14

14

Operates like a timer thattriggers the events in a setsequence

The control of cell cycle

The system of switches is binary(ON/OFF) and launches events ina complete, irreversible fashion

The control system isindependent of the events itcontrols

The system is highly adaptable andcan be modified to suit specific celltypes

15

15

The Cyclin-Cdk complexes: the majorcomponents of the cell cycle control system

16

16

Cyclically activated protein kinases control cellcycle progression

Cyclin-dependent kinases (Cdks)

Kinase’s activity oscillates in the cell cycle

Cyclical changes in Cdk activity are controlled byan array of enzymes and other proteins

Among these, cyclins are the majormolecules

The “go”system (or the “engine”)

Expression is constant through the cellcycle

17

17

The cyclins

Four classes of cyclins:1. G1-cyclins—help to promote passage through“Start”or the restriction point in late G1

2. G1/S-cyclins—bind Cdks at the end of G1 andcommit the cell to DNA replication

3. S-cyclins–binds Cdks during S phase and arerequired for the initiation of DNA replication

4. M-cyclins—promote the events of mitosis

18

18

The major Cyclin-CDK complexes

19

19

Regulation of cell cycle

20

20

Regulation of the cyclin-Cdk complex

Post-translational modification

Transcriptional regulation

Cyclical proteolysis

Inhibitors of Cdks

21

21

Activation of the cyclin-Cdk complex

Partial activation of Cdks by cyclin associationFull activation by Cdk-activating kinase (CAK) phosphorylation

Active sitehindered byinhibitory T-loop

Exposure of T-loop by cyclinbinding

Phosphorylation byCAK causesconformationchange

Study on crystal structure of CDK2 revealed:

22

22

Regulation of the cyclin-Cdk complex:post translational modification

Inhibition of Cdks by inhibitory phosphorylation

Inhibition: Phosphorylation by Wee1 kinase at Tyr15Cdc25 phosphatase dephosphorylates the p-Tyr15 andactivates the Cdk

Human Cdk1 and also Cdc2 in yeast:

PP2A

23

23

The inhibition of a cyclin-Cdk complex by Cdk inhibitorproteins (CKI)

CKI (Cdk inhibitor proteins):

The INK family, P15, p16, p18, and p19 target to CDK4, 6

The CIP/KIP family, p21, p27, and p57 target to mostCDKs

Binding of CKI renders Cdk inactive by conformation changeand rearranged structure of active site

(during G1)

(during all phases)

24

24

Cyclical proteolysis regulates cyclin-Cdk activity

SCF complex(Ubiquitin ligase)

Regulated by SCF complex in G1 and S phase

Targets:G1/S cyclins, some CKIs (such as p27Kip)

25

25

(Ubiquitin ligase)APC complex

Cyclical proteolysis regulates cyclin-Cdk activityRegulated by APC complex in M phase

APC: anaphase promoting complex

Target: M cyclin

26

26

The G1, G1-S, and S phase

27

27

The G1 phase is a state of stable Cdk inactivity

To reach a stable G1 for growth, Cdk reactivation is prevented

Decline in M-cyclin and in Cdc20-APC

Increase in Cdh1-APC* activitywhich targets to M-cyclins

*Cdh1 is a close relative of Cdc20

Sic1, a Cdk inhibitor ( CKI),inactivates M-Cdk

Decrease in M-cyclin transcription

*Cdh1-APC targets to M-cyclins to inactivate M-Cdk after mitosis

28

28

Initiation of S phase in budding yeast

Accumulation of G1-cyclin that is resistant to Sic-1 and Hct-1-APC

G1-Cdk stimulates G1/S-cyclin synthesis

G1/S-Cdk stimulates S-cyclin synthesis, and increase in S-Cdk activity

G1/S-Cdk phosphorylates Sic1 and Hct-1 and blocks their activity

29

29

Initiation of S phase in animal cellsIn G1, cells are in a state of Hct-1 activation, accumulation of aCKI, and inhibition of cyclin gene expression (M-cyclin)

In late G1, activation of G1-Cdk reverses the inhibitory state of G1

G1-Cdk and G1/S-Cdk phosphorylate Rb, an inhibitor of cellcycle progression, and results in release of E2F

30

30

Mitogens stimulate activation of Ras and a MAPkinase cascade to trigger cell division

31

31

Mechanisms of Myc-promoted cell cycle entry

32

32

Figure 17-23 Molecular Biology of the Cell (© Garland Science 2008)

Control of the initiation for DNA replication

(formed by Orc proteins)

33

33

The control for DNA replication

Regulation of pre-replicative complex (pre-RC) by S-CDK

ORC: Origin recognition complexBinds to the replication origin through the cell cycle

Cdc6, Cdt1 associate with ORC at early G1

help to recruit Mcm proteins, a DNA helicase

S-Cdk causes:

Control only one replication, by Cdc6 degradation andby Mcm export from nucleus

Start of DNA replication, by ORC phosphorylation,MCM activation, and replication firing

34

34

The M phase:Mitosis and cytokinesis

35

35

Regulation of entry into mitosis

36

36

• M-cyclin accumulates by reduced degradation before M phasein embryonic cell cycles

• M-cyclin increases in transcription in most cell types

• Inactivated M-cyclin-Cdk complex accumulates due toinhibitory phosphorylation

• Cdc25 phosphatase dephosphorylates and activates M-Cdk

• Polo kinase and active M-Cdk activate Cdc25

• Active M-Cdk inactivates Wee1

Entry into mitosis

37

37

Multiple roles of M-Cdk in mitosis

Induce the assembly of mitotic spindle

Ensure replicated chromosomes attach to the mitoticspindle

Chromosome condensation

Nuclear envelope breakdown

Reorganization of the Golgi apparatus and endoplasmicreticulum

38

38

Mitosis

39

39

Mitosis, continued

Exit

40

40

Sister chromatid separation is triggered by proteolysis

Separase is required forsister chromatids separation

Securin, can inactivateseparase; degradation ispromoted by APC

APC: anaphase-promotingcomplex, a highly regulatedubiquitin ligase; promote thedestruction of several mitoticregulatory proteins

Cdc20 activates APC

41

41

The spindle-attachment checkpoint

Ensure that all chromosomes are properly attached tothe spindle before sister-chromatid separation occurs

A sensor mechanism monitors the state of thekinetochore, the specialized region of thechromosome that attaches to microtubules of thespindle

Improper attachment of kinetochore to the spindlesends out a negative signal to the cell-cycle controlsystem, blocking Cdc20-APC activation and sisterchromatid separation

Several proteins, including Mad2, are recruited tounattached kinetochores. Mad2 binding results in Inhibitionof Cdc20-APC and blocking securin destruction

42

42

Exit from mitosis and start of G1

The mitotic spindle must be dissembled

Complex changes at the end of mitosis

Chromosomes decondensedThe nuclear envelope reformed

Inactivation of M-Cdk is required for exit from mitosis

Cdc20-APC complex mediated ubiquitin-dependentproteolysis of M-cyclin

43

43

Decline in M-cyclin and in Cdc20-APC

Increase in Cdh1-APC* activitywhich targets to M-cyclins

*Cdh1 is a close relative of Cdc20

Sic1, a Cdk inhibitor ( CKI),inactivates M-Cdk

Decrease in M-cyclin transcription

*Cdh1-APC targets to M-cyclins to inactivate M-Cdk after mitosis

Exit from the M phase and entry ofG1 phase in somatic cells

44

44

DNA damage checkpoints and p53 activation

Normally, P53 is targeted forubiquitin-dependentdegradation by Mdm2, aubiquitin ligase

p53 is phosphorylated byDNA damage-activatedkinase and is released frombinding to Mdm2

45

45

An overview of the cell-cycle control system

Accelerators (cyclin-CDK) and brakes (checkpoints)

46

46

From cell dividing to senescence

-gal-positiveFlat morphology

Abnormal nuclear morphology

47

47

In 1965, Leonard Hayflick first suggested thatthere was a “finite limit to the cultivation periodof diploid cell strains”and this was“attributable to intrinsic factors which areexpressed as senescence at the cellular level”.

Hayflick proposed that normal cells cannot divideindefinitely because they are programmed for aset proliferative lifespan.

Cell senescence observed by Hayflick

48

48

Cellular senescence:A state of irreversible growth arrest

Can be triggered by multiple mechanismssuch as: telomere shortening, DNA damage, and INK4a/ARFderepression

Cell 130, 223-233, 2007

49

49

Nature Reviews Cancer 1:203, 2001

Telomere

Molecular Cell Biology, 6th edition

GGGTTA repeats (near thousands) in human’s telomere

50

50

Telomere shortening and senescence

Human diploid fibroblasts (HDFs) undergo 60-80doublings in culture, after which they cease divisionand develop a senescent phenotype

Telomerase expression in HDFs is sufficient tobypass the cell senescence and crisis

Nature Reviews Cancer 1:203, 2001

51

51

The telomere problem of a linear DNAreplication

The “must be”5’to 3’polymerization, the lagging stand, and the telomere

52

52

Maintaining telomere length immortalizes cells

Nature Reviews Cancer 1:203, 2001

Exogenous expression of telomerase reverse transcriptase (TERT)at any stage in the replicative period allows immortalization of thesecells

53

53

Telomere hypothesis

• Most adult cells have limiting amounts of telomerase that arenot sufficient for preventing telomere loss, resulting in theshortening of telomeres with ages

• Telomerase-deficient mice with short telomeres show reducedfunction of various stem cell compartments including those inthe bone marrow and skin

• Telomerase-deficient mice show a decrease in both the medianand maximum lifespan, and these effects become morepronounced with each subsequent generation

• Terc transgenic mice show increased mortality due to cancer,but show evidence improved tissue regeneration as well as aslight increase in maximum life span

Nature 2007, 448:767-774

54

54

Telomere stabilization: an important step in tumordevelopment

Most human cells don’t express telomerase, whereas mosthuman tumors express telomerase

Expression of dominant-negative telomerase reversetranscriptase (TERT) results in apoptosis of tumor cells

Those tumors that don’t express telomerase maintain thetelomere by an mechanism of alternative lengthening oftelomeres (ALT)

55

55

Age-dependent increase in the number of senescent cellsin differentiated tissues

56

56

Genomic instability in aging cells

DNA damage and mutations accumulate with agein mammals

Cytogenetically visible lesions such as translocations,insertions, dicentrics, and acentric fragmentsaccumulate in aging mammalian cells

57

57

DNA damage

DNA repair

Genomic instability

Recovery and regenerate

Defect in DNA repair

Aging?

The role of DNA repair in aging

58

58

Genomic instability increased not only in agingcells but also in most cancer cells

59

59

Excessive oncogenic induction can result in cell-cyclearrest or apoptosis

60

60

OIS: Oncogene-induced senescence

ARF-p53 pathway

p16INK4a-RB pathway

Implication: in response to the activation of mitogeniconcogenes, checkpoint-mediated failsafe mechanismssuch as apoptosis or cellular senescence may berecruited to terminate a pre-malignant condition

Trends cell biol 11, s27-s31, 2001

Nature Rev Cancer 3, 286-295, 2003

61

61

Pathways to senescence

Nature Reviews Cancer 1:203, 2001

62

62

• Please introduce some key background information regarding thispaper, including the motivation or hypotheses for pursuing this study,cdk2, myc, relationship between cdk2 and myc, oncogene-inducedsenescence (such as Ras), etc.

• What has been found in this paper is different from the finding inprevious studies regarding myc activity in cell proliferation andapoptosis?

• Why authors think myc-induced senescence in the absence of cdk2 isnot due to blocking apoptosis? And myc-induced cell proliferation isindependent of cdk2?

• Why authors conclude that Arf-p53-p21 and p16-pRb pathways wereintegrally required for myc-induced senescence in cdk2-/- cells?

Assignments for next week’s class

Cdk2 suppresses cellular senescence induced by the c-myc oncogene,Nat Cell Biol. 2010 Jan;12(1):54-9

63

63

• Did authors conclude that cdk2 regulates myc-induced senescencethrough controlling myc-induced genotoxic stresses? Why?

• Why oxidative stress may contribute to myc-induced senescence?What is the role of cdk2 in this?

• Since CDK2 is dispensable in many cancer cell lines, why authorsstill think inhibition of CDK2 can be a therapeutic consideration fortreatment of cancer?

Assignments for next week’s class, continued,