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基醫所

Cell Cycle
Regulation and Checkpoints

Chi-Wu Chiang, Ph
...

IMM, NCKU
1

1

From cell division to a full grown body
-----Accompanied with cell division, cell growth and cell death

cell mass
Cell mass=cell number+cell size

2

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Cell division, cell growth, and cell death are
independently regulated but linked
Intrinsic programs
Regulated cell cycle progression
Programmed cell death
Extracellular signal molecules
Mitogens
Growth factors
Survival factors
Apoptosis-inducing factors
Proliferation inhibitors
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Introduction to cell cycle and cell cycle
checkpoints

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Cell cycle
A cell reproduces by performing an orderly sequence of events in which
it duplicates its contents and then divides in two

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The phases of cell cycle

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Embryonic cell cycle and somatic cell cycle
Somatic cell cycle

30 min

6h
Early embryonic cell cycle

M
G1

G2

12h

M
S

S

6h
7

7

Yeast provides a good genetic system for study
on cell cycle
Mostly proliferating in a haploid state
Easy for genetic manipulation
Cdc genes and cdc mutants ----cell-division-cycle genes

8

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Cdc mutants selected by temperature
sensitive mutations
A mutant that cannot complete the cell cycle, cannot be
propagated
Temperature-sensitive mutants
Functioning in permissive condition (low temp
...
)

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Xenopus oocyte: a giant cell for study cell cycle
biochemically
1 mm in diameter
Carrying 100,000 times more cytoplasm than an average cell in the
human body

After fertilization occurs, the first division takes about 90 minutes,
and the next 11 divisions occur at 30-mim intervals, producing
about 4096 cells within 7 hours
Each cycle is divided into S and M phase without detectable G1 or G2
phases
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Studying the cell cycle in a cell-free system
Observation of repeated nuclei decondense and DNA replication and mitosis
In vitro

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Measurements of cell cycle progression
Propidium iodide (PI) staining

EMBO 2003, 22:5459

BrdU
staining
(bromodeoxyuridine)

PI
staining
12
Oncogene 2001, 20:4507

12

The cell-cycle control system

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The control of cell cycle
Operates like a timer that
triggers the events in a set
sequence
The system of switches is binary
(ON/OFF) and launches events in
a complete, irreversible fashion
The control system is
independent of the events it
controls
The system is highly adaptable and
can be modified to suit specific cell
types
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The Cyclin-Cdk complexes: the major
components of the cell cycle control system

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Cyclically activated protein kinases control cell
cycle progression
Cyclin-dependent kinases (Cdks)The “ system (or the “
go”
engine”
)
Expression is constant through the cell
cycle
Kinase’ activity oscillates in the cell cycle
s
Cyclical changes in Cdk activity are controlled by
an array of enzymes and other proteins
Among these, cyclins are the major
molecules
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The cyclins
Four classes of cyclins:
1
...
G1/S-cyclins—bind Cdks at the end of G1 and
commit the cell to DNA replication
3
...
M-cyclins—promote the events of mitosis
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The major Cyclin-CDK complexes

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Regulation of cell cycle

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Regulation of the cyclin-Cdk complex
Post-translational modification
Transcriptional regulation
Cyclical proteolysis
Inhibitors of Cdks

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Activation of the cyclin-Cdk complex
Partial activation of Cdks by cyclin association
Full activation by Cdk-activating kinase (CAK) phosphorylation
Study on crystal structure of CDK2 revealed:

Active site
hindered by
inhibitory T-loop

Exposure of Tloop by cyclin
binding

Phosphorylation by
CAK causes
conformation
change
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Regulation of the cyclin-Cdk complex
:post translational modification
Inhibition of Cdks by inhibitory phosphorylation
Human Cdk1 and also Cdc2 in yeast:
Inhibition: Phosphorylation by Wee1 kinase at Tyr15
Cdc25 phosphatase dephosphorylates the p-Tyr15 and
activates the Cdk

PP2A
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The inhibition of a cyclin-Cdk complex by Cdk inhibitor
proteins (CKI)
CKI (Cdk inhibitor proteins):
(during G1) The INK family, P15, p16, p18, and p19 target to CDK4, 6
(during all phases) The CIP/KIP family, p21, p27, and p57 target to most
CDKs
Binding of CKI renders Cdk inactive by conformation change
and rearranged structure of active site

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Cyclical proteolysis regulates cyclin-Cdk activity
Regulated by SCF complex in G1 and S phase
SCF complex(Ubiquitin ligase)
Targets:G1/S cyclins, some CKIs (such as p27Kip)

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Cyclical proteolysis regulates cyclin-Cdk activity
Regulated by APC complex in M phase
APC: anaphase promoting complex
APC complex (Ubiquitin ligase)
Target: M cyclin

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The G1, G1-S, and S phase

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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
Decrease in M-cyclin transcription
Increase in Cdh1-APC* activity
which targets to M-cyclins
Sic1, a Cdk inhibitor ( CKI),
inactivates M-Cdk
*Cdh1 is a close relative of Cdc20
*Cdh1-APC targets to M-cyclins to inactivate M-Cdk after mitosis
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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
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Initiation of S phase in animal cells
In G1, cells are in a state of Hct-1 activation, accumulation of a
CKI, 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 cell
cycle progression, and results in release of E2F

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

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Mechanisms of Myc-promoted cell cycle entry

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Control of the initiation for DNA replication
(formed by Orc proteins)

32
Figure 17-23 Molecular Biology of the Cell (© Garland Science 2008)

32

The control for DNA replication
Regulation of pre-replicative complex (pre-RC) by S-CDK
ORC: Origin recognition complex
Binds 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:
Start of DNA replication, by ORC phosphorylation,
MCM activation, and replication firing

Control only one replication, by Cdc6 degradation and
by Mcm export from nucleus
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The M phase
:Mitosis and cytokinesis

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Regulation of entry into mitosis

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Entry into mitosis
• M-cyclin accumulates by reduced degradation before M phase
in embryonic cell cycles
• M-cyclin increases in transcription in most cell types
• Inactivated M-cyclin-Cdk complex accumulates due to
inhibitory phosphorylation
• Cdc25 phosphatase dephosphorylates and activates M-Cdk
• Polo kinase and active M-Cdk activate Cdc25
• Active M-Cdk inactivates Wee1

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Multiple roles of M-Cdk in mitosis
Induce the assembly of mitotic spindle
Ensure replicated chromosomes attach to the mitotic
spindle
Chromosome condensation
Nuclear envelope breakdown
Reorganization of the Golgi apparatus and endoplasmic
reticulum
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Mitosis

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Mitosis, continued

Exit

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Sister chromatid separation is triggered by proteolysis
Separase is required for
sister chromatids separation
Securin, can inactivate
separase; degradation is
promoted by APC
APC: anaphase-promoting
complex, a highly regulated
ubiquitin ligase; promote the
destruction of several mitotic
regulatory proteins
Cdc20 activates APC
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The spindle-attachment checkpoint
Ensure that all chromosomes are properly attached to
the spindle before sister-chromatid separation occurs
A sensor mechanism monitors the state of the
kinetochore, the specialized region of the
chromosome that attaches to microtubules of the
spindle
Improper attachment of kinetochore to the spindle
sends out a negative signal to the cell-cycle control
system, blocking Cdc20-APC activation and sister
chromatid separation
Several proteins, including Mad2, are recruited to
unattached kinetochores
...

Hayflick proposed that normal cells cannot divide
indefinitely because they are programmed for a
set proliferative lifespan
...
2010 Jan;12(1):54-9
• Please introduce some key background information regarding this
paper, including the motivation or hypotheses for pursuing this study,
cdk2, myc, relationship between cdk2 and myc, oncogene-induced
senescence (such as Ras), etc

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