Cell Cycle and Cell Growth Control

By Chung-Yih Wang, MD, PhD

chungyihwang@yahoo.com.tw

National Taiwan University Medical SchoolUniversity of Wisconsin-MadisonUniversity of Michigan, Ann ArborUniversity of ChicagoNational Yan-Ming UniversityVeterans General Hospital-Taipei, Cancer CenterCheng-Hsin Hospital, Department of Radiotherapy

Course Organization

Cell cycle controlMitosis Apoptosis Cancer Biology and Novel Treatment

Strategy Against Cancer

Learn Goals

Understand the stages of the cell cycle Key Factors controlling

the passage of the cell cycle The role of apoptosis in

physiology/ Pathology Controls of apoptosis

Carcinogenesis Oncogenes and tumor

suppressor genes New strategies for

cancer therapy

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 Streptomyceshygroscopicus [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 immunosuppressiveand antiproliferativeproperties

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

12/26

Human

12/3123:59:58

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

Saccharomycescerevisae Budding Yeast

Schizosaccharomycespombe 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

Cell Fusion Experiment

M+ G1 M+S M+G2

Cell Fusion Experiment

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 un-fertilized 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 Xenopusoocyte, 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 cyclin1.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 M

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 cyclingene transcriptionWhat 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 S-

Cyclin 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 phosphorylationby wee1

Activation of Cdc25 phosphotase leads to activation of M-Cdk, which then positively enhance its own activation

Entry into Mitosis