Cell Cycle and Cell Growth Control-2011 Text

Cell Cycle and Cell Growth
Control
By
Chung-Yih Wang, MD, PhD
chungyihwang@yahoo.com.tw

National Taiwan University Medical School
University of Wisconsin-Madison
University of Michigan, Ann Arbor
University of Chicago
National Yan-Ming University
Veterans General Hospital-Taipei, Cancer Center
Cheng-Hsin Hospital, Department of Radiotherapy
Course Organization
Cell cycle control
Mitosis
Apoptosis
Cancer Biology and Novel Treatment
Strategy Against Cancer
Learn Goals
Understand the stages
Carcinogenesis
of the cell cycle
Oncogenes and tumor
Key Factors controlling
suppressor genes
the passage of the cell
New strategies for
cycle
cancer therapy
The role of apoptosis in
physiology/ Pathology
Controls of apoptosis
Examination
A thorough understanding of the
physiological processes and mechanisms is
more important than memorizing details and
terminology
15 multiple choice questions and two to three
The grades will be adjusted to match normal

distribution
Why bother learning the basic medical

science?
Anti-fungus treatment
Organ Transplantation
Cancer Treatment
Autism
Rapamycin
Rapamycin was first
discovered as a product of
the bacterium Streptomyces
hygroscopicus [1] in a soil
sample from Easter Island
originally developed as an
antifungal agent
this was abandoned when it
was discovered that it had
potent immunosuppressive
and antiproliferative
properties
mTOR: mammalian
Target of Rapamycin
Rapamycin
Organ Transplantation
used in coronary stents to prevent re-stenosis
following balloon angioplasty
Cancer treatment
Potential treatment for autism
Reductionism
Biology is a
reductionistic Science
To understand the
nature of complex
things by reducing
them to simpler or
more fundamental
things
Holism
the idea that all the properties of a given
system cannot be determined or explained by
its component parts alone. Instead, the
system as a whole determines in an important
way how the parts behave.
Aristole: Metaphysics: "The whole is
different from the sum of its parts"
Life History on Earth
Birth of Earth
1/1
Birth of Life
2/17
Eukaryotic Life
9/5
Mammal
Human
12/26 12/31
23:59:58
Cell Cycle
What You Need to Know?

The stages and their physiological roles of
cell cycle ( G1, S, G2, M)
Key regulators and their mechanisms of
regulating cell cycle progression (Cyclin,
CDK, CKI..)
Cell Cycle
Cell cycle is the series of events that take place in a
dividing cell.
These events can be divided in two main parts:
Inter-phase (G1, S, and G2) and M-phase
The molecular events that control the cell cycle are
ordered and directional; that is, each process occurs
in a sequential fashion and it is impossible to
"reverse" the cycle.
Leland H. Hartwell, R. Timothy Hunt, and

Paul M. Nurse won the 2001 Nobel Prize in
Physiology or Medicine for their discovery
of the central molecules in the regulation of
the cell cycle.
Cell Cycle
Overview of Cell Cycle
Techniques and Materials

Commonly Used in The
Study of Cell Cycle
Xenopus laevis (African

Clawed Frog)
Xenopus laevis
Large size
Huge amount of cytoplasm
Rapid, synchronously
division after fertilization
Easy to manipulate
cytoplasm and nucleus
S and M with no
interference from G1 or G2
control mechanisms
Yeast
Saccharomyces
cerevisae
Budding Yeast
Schizosaccharomyces
pombe
Fission yeast
Yeast
Rapid growth
Small genome size (1%
that of mammalian)
Easy to manipulate
genetically
Can proliferate as
haploid
Easy to isolate and

study mutant with
phenotype in cell cycle
Cell-division-cycle
genes (cdc)
Conditional mutation
Well conserved cell
cycle control genes
from yeast to human
Cells
3 Categories of Cells
Can not divide: Terminally
differentiated cells: Nerve
cells, Muscle cells, Red
blood cells
Normally do not divide:
Liver cells, Mature
Lymphocytes
Normally keep dividing:
Fertilized egg, Bone
marrow, Epithelium
Control of Cell Cycle Progression
Cell Cycle
Cell Fusion Experiment

In the 1970
Whether the cytoplasm
contains the regulatory
factors that affect cell cycle
activities
Cytoplasm of a replicating
cell contains factors that
stimulate DNA synthesis
M+ G1
M+S
M+G2

The transitions from
G1 to S and from G2 to
M are both under
positive control
Both are induced by
stimulatory factor(s)
What is the factor(s) that promotes the

transitions from G1 to S and from G2 to M ?
Isolation of MPF
Maturation
Promoting
Factor : MPF
MPF activity is also

present in mammalian cells,
Hela, and sea urchin eggs
Sea urchin eggs fail to
undergo mitosis if put in
protein synthesis inhibitor:
MPF require new protein
synthesis
Fluctuation of the protein
during cell cycle: Cyclin
MPF
1983 Tim Hunt et, al

use 35S methionine to
label protein in
fertilized and unfertilized sea urchin
egg.
What will you expect
the MPF to behave in
such experiment?
MPF
Protein synthesis inhibitor
blocked MPF activity
35S Methionine labelling
followed by
electrophoresis identify
possible candidates for
MPF
The potential MPF will
appear and disappear at
specific time
corresponding to cell cycle
MPF
Partially purified MPF
stimulate the
incorporation of 32P
into proteins
Suggesting that MPF is
a protein kinase
Purified MPF contains
two subunits, 32kDa
and 45 kDa
cDNA of putative
cyclin were cloned
mRNA of cyclin, when
injected to Xenopus
oocyte, can promote
cell cycle progression
Yeast cdc
Ts
Cell-division-cycle genes
(cdc)
MPF/Yeast cdc
Yeast cdc2 gene
encode a 34kDa
protein kinase p34cdc2
Antibody against cdc2
react with 32kDa
protein of MPF
Vertebrate cdc2
homologue dose not
fluctuate during the
cell cycle, which
suggest that this 32kDa
kinase depend on
another protein
45 kDa : Cyclin
MPF
An activities identified in frog
oocytes that promote
mitosis
MPF activity fluctuate
during the cell cycle
Two components, one
needs novel protein
synthesis: Cyclin, the other
one contains kinase activity:
CDK
Human, Frog, Sea

urchin, and yeast are
all close relatives
MPF
Consist of two subunits

One regulatory subunit:Cyclin
One subunit with kinase activity: Cdk
Fission yeast
The same Cdk (cdc2) is
responsible for G1 and
M
Mammalian Cell Cycle

There are four classes of
cyclins
Each cyclin binds to and
regulate the activity of a
Cdk
G1/S Cyclin: commit the
cell to DNA replication
S Cyclin: Initiation of
DNA replication
M Cyclin: mitosis
G1 Cyclin: Start
How is CDK Regulated

During Cell Cycle?
Cyclin Regulates Cdk

Binding of cyclin
1.causes a conformation
change and activation
of Cdk
2. Directs it to specific
target
Wee1 encodes a
tyrosine kinase
Cdc25 encodes a
phosphotase
Regulation of CDK in yeast
Phosphorylation
Regulation of CDK
Cyclin Binding
Cdk Phosphorylation

Inhibitory
Stimulatory
Cdk Inhibitors
Controlled Proteolysis
Subcellualr Localization
Cdk Inhibitory Proteins (CKIs)

P21 , P27
Crucial In the
pathogenesis of Cancer
Proteolysis Control (G1, S)

Cyclin and CKI
destruction by a
Ubiquitin-conjugating
system
G1/S: SCF
Ubiquitylation by SCF is
controlled by
phosphrylation, only
specifically
phosphorylated proteins
are recognized
Proteolysis Control (M)
Subcellular Localization
(Cyclin B1, M-cyclin)
G2
Cdk Regulation
Cyclin Binding
Cdk Phosphorylation

Inhibitory
Stimulatory
Controlled Proteolysis
Cdk Inhibitors
Subcellualr Localization
S Phase Initiation
How does a cell know
when to start DNA
synthesis
CDK activity in G1 is suppressed by:

accumulation of CKIs, inhibition of cyclin
gene transcription
What control the transcription of cyclin gene?
Rb in G1-S
Transcription of G1/S
and S-Cyclin requires
E2F
E2F is inhibited by
binding with pRb
Retinoblastoma (Rb) is a rapidly developing

cancer which develops in the cells of retina .
A very treatable cancer
There are two forms of the disease, heritable
form and a non-heritable form.
S Phase Initiation
Slow accumulation of G1-Cdk
during G1
G1-Cdk phosphorylates pRb
E2F enhance its own and SCyclin transcription
S-Cdk increases pRb
phosphorylation
S-Cdk phosphorylates and
inactivate P27-CKI
E2F promote transcription of
genes required for DNA
synthesis
S Phase Initiation
Result:
Rapid and Complete activation of S-Cdk and
S-initiation
A typical example of biological control
system with multiple feedback loop
Checkpoint
Cell cycle progress will
stop if:
DNA is damaged
DNA is not properly
replicated during S
Chromosomes are not well
aligned during M
Checkpoints ensure the
integrity of genome during
cell cycle
DNA Damage Chcekpoints

G1:
Phosphorylation and
activation of of P53 ,
Transcription
activation of P21
G2
Phosphorylation and
destruction of CDC25
G2CDK remains
inactive without
CDC25
G1 DNA Damage Checkpoint
ATM (Ataxia-telangiectasia Mutation)

Story of Ataxia-telangiectasia

a rare, neurodegenerative, inherited disease

affects the cerebellum, immune system and
predispose to cancer
increased sensitivity to ionizing radiation, but
normal response to other forms of radiation such
as ultraviolet light
Entry into Mitosis

M-cyclin transcription
increase during G2 and M
Gradual accumulation of
M-Cdk, which are
phosphrylated by CAK, but
remain inactive due to
inhibitory phosphorylation
by wee1
Activation of Cdc25
phosphotase leads to
activation of M-Cdk,
which then positively
enhance its own
activation
Entry into Mitosis

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Cell Cycle and Cell Growth

The grades will be adjusted to match normal

Why bother learning the basic medical

Life History on Earth

What You Need to Know?

Leland H. Hartwell, R. Timothy Hunt, and

Overview of Cell Cycle

Techniques and Materials

Xenopus laevis (African

Easy to isolate and

Control of Cell Cycle Progression

Cell Fusion Experiment

Cell Fusion Experiment

Cell Fusion Experiment

What is the factor(s) that promotes the

MPF activity is also

1983 Tim Hunt et, al

Human, Frog, Sea

Consist of two subunits

Mammalian Cell Cycle

How is CDK Regulated

Cyclin Regulates Cdk

Regulation of CDK in yeast

Cdk Inhibitory Proteins (CKIs)

Proteolysis Control (G1, S)

Proteolysis Control (M)

CDK activity in G1 is suppressed by:

Retinoblastoma (Rb) is a rapidly developing

DNA Damage Chcekpoints

G1 DNA Damage Checkpoint

ATM (Ataxia-telangiectasia Mutation)

a rare, neurodegenerative, inherited disease

Entry into Mitosis

Entry into Mitosis

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