Lecture 17Regulation of the Cell Cycle

and Cell Death

The Cell Division Cycle

A cell contains all the information necessary for making a copy of itself during cell division

Cells reproduce by carrying out a highly ordered sequence of events called the CELL CYCLE

– Typically Involves:Cell GrowthDNA ReplicationDistribution of Duplicated Chromosomes to Daughter CellsCell Division

Phases of the Eukaryotic Cell Cycle

Different Cell Types have Variations in Timing, Control and Progression Through Cell Cycle

In the Adult Human Body

-Most Cells Are Not Actively Dividing

-Some Cells Are Continuously Dividing- Example: Bone Marrow Cells, Epithelial Cells

Some Cells Do Not Divide After Differentiation

Example: Nerve Cells

Some Cells can Be Triggered to Divide to Replace Cells that have Died

Example: Cells of Liver Tissue

Control of Cell Cycle Involves Monitoring

Intracellular And Extracellular Conditions

The Control System Needs to Regulate Progression through the Cell Cycle to Ensure:

1)Events Associated with Each Phase are Carried Out at the Appropriate Time and in the Appropriate Order

2) Each phase is complete Before Next Phase is Initiated

3) Must be Able to Respond to External Conditions Required for Cell Growth and Division.

A Series of Control Points called Checkpoints Accomplish These Objectives

Cell Cycle Checkpoints Regulate Progression Through the Cell Cycle

Cell Fusion Experiments Provide Evidence for Control Molecules in the Cell Cycle

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S

S S M M

MG1 G1

S Phase is ActivatedIn Original G1 Nucleus

M phase is ActivatedIn Original Non-M Nucleus

The Cell Cycle Is Controlled by Cyclin Dependent Kinases (CDK)

CDK forms a Complex with a Protein called a Cyclin to form a CDK Complex

Different Cyclin-CDK Complexes Trigger Different Steps in the Cell Cycle

4 Major Types of Cyclin-CDK Complexes: G1-CDKG1/S-CDKS-CDK M-CDK

Regulation of the Activity of CDK Complexes

1) Proteolysis of Cyclin Component

2) Phosphorylation/Dephosphorylation

3) Binding of Inhibitory Proteins

4) Subcellular Localization

Cyclin Protein Concentration and Cyclin-CDK Activity Oscillate during the Cell Cycle

Post Transcritional Control of Cyclins:M-Cyclin is Ubiquitinated and Subsequently

Degraded by the Proteasome

Cyclin Destruction Occurs at Specific Points during the Cell Cycle

Checkpoints in the Cell Cycle

Progression through the G1 Checkpoint

Cells Need to Check for : Cell SizeNutrientsMitogens and Growth Factors DNA Damage

Called the Restriction Point in MammalsCommits Cell to the Process of Cell Division

G1 checkpoint

G1G1

G0

Cells Can Delay Cell Division by Entering Specialized Nondividing State, GO

Green Light to Proceed: Environment Favorable

Red Light – Don’t ProceedEnvironment Unfavorable

(Absence of Mitogenic Signals)

Most cells in our body are In Go State:

In the Absence of Mitogenic Signals,

The protein Rb Inhibits Cell Cycle Progression Rb- Retinoblastoma Protein

Binds Transcription Factor E2F and Prevents Function

E2F Required for:Activating TranscriptionOf Genes Encoding ProteinsRequired for G1/S transition

G1/S Cyclins, S Cyclins andComponents of DNA

Replication Machinery

The G1-CDK Complex Controls the G1 Checkpoint by Phosphorylating the Rb Protein

G1 DNA Damage Checkpoint

DNA Damage Needs to be Repaired before DNA Synthesis Occurs

Activation of Transcription Factor p53 leads to synthesis of CDK inhibitor (CKI), p21

p21 binds and inhibits G1/S CDK and S-CDK Complexes

Cells Arrest Until Damage is Repaired and Then Progress

Or

Undergo Apoptosis if Unable to Repair

p53 Mediates the G1 DNA Damage Checkpoint Through Inactivation of G1/S- and S-CDK Complexes

Checkpoints in the Cell Cycle

S-CDK Complexes Are Required for DNA Replication During S-phase

S-CDK Controls:1) Initiation of Replication 2) Prevents Re-replication from a Particular Origin

Progression Through the G2 Checkpoint to Enter Mitosis

Check for:Cell SizeDNA Replication Complete

Passage through G2 Checkpoint Requires Active M-CDK:

Functions to Phosphorylate Proteins Involved in Early Stages of Mitosis

1) Nuclear Envelope Breakdown2) Chromosome Condensation3) Mitotic Spindle Formation 4) Targeted Protein Degradation

Unreplicated DNA SensedBlocks Activating

Phosphatase

Unreplicated DNA Blocks Activation of M-CDK Complex

T

Checkpoints in the Cell Cycle

M-CDK Controls The Spindle Assembly Checkpoint by

Activating the Anaphase Promoting Complex (APC)

Check for:

Proper Chromosome Attachment to Spindle

Phosphorylation of APC- now Activated

Sister Chromatids Can Separate

If Chromosomes not Properly Attached

Metaphase Arrest

Will not Separate

Exit From Mitosis

Now Need to Reverse Events:Nuclear Envelope BreakdownChromosome CondensationMitotic Spindle Formation

Destroy M-CyclinM-CDK Activates APC

APC Targets M-Cyclin for Destruction by the Proteasome

Cancer Involves Defective Cell Cycle Control Mechanisms

Cancer Inducing Mutations Inherited or Introduced by Viruses

Two Key Types of Mutated Genes That Can Lead to Cancer:

Oncogenes - Gene whose presence can trigger development of cancer.

Example: ras, bcl-2 Tumor Suppressors- Gene whose absence or

inactivation can lead to cancerExample: p53, Rb

Mitogens and Growth Factors Activate the Ras Pathway Leads to Passage through the G1 Checkpoint

p53 Can Induce Apoptosis in

DNA Damaged Cells

Can’t RepairDNA DAMAGE

Apoptosis

Apoptosis:Regulated Cell Death

Role in Killing of Unneeded, Damaged, or Potentially Deleterious Cells

Occurs in Embryonic and Adult Tissues

Proteins Involved are Always Present in Cells- Needs to Be Activated by Stimuli

Can Result From:

Developmental Cues

Withdrawl of Essential Growth Factors

DNA Damage

Various Cell Stresses

Programmed Cell Death

• Cell Death Occurring at a Defined Point in Development

• Usually proceeds by Apoptosis

Mouse Paws

Not All Cell Death is Apoptotic

Apoptosis:

An Active Regulated Process

DNA FragmentationChromatin Condensation

Fragmentation of NucleusCell Shrinks

Formation of Membrane Enclosed Fragments called Apoptotic Bodies

Recognition and Engulfment by Phagocytic Cells

or Neighboring Cells

Oncosis and Necrosis:

Unregulated Cell Death Due to Injury

Cell Swells (Oncosis)

Nucleus Swells

Disruption of Organelles and Rupture/Release of Contents

Contents Released into Extracellular Space

Necrosis and Apoptosis are Morphologically Distinct

Necrosis Apoptosis

The Morphological Changes of Apoptosis Are Orchestrated by Caspases

Cysteine Proteases that cleave at Aspartic Acid Residues

Activate Apoptosis by Cleaving Specific SubstratesPresent but inactive in cells

Two Main Types of Caspases1) Initiators- Need to dimerize to become active “induced proximity”

2) Executioners- Need to be proteolytically cleaved to become active- Cleavage is usually Mediated by Initiator Caspases

Once Executioners are Activated their Key Targets of Proteolysis Include:

1)An Inhibitor of a DNAse- Fragmentation of DNA

2)Nuclear Lamins- Fragmentation of Nucleus

3)Other Cytoskeletal Associated Proteins- Disruption of Cytoskeleton and Cell Fragmentation

4)Activation of Additional Caspases

Caspases are Present but Inactive in Cells

Zymogens: Proteins initially synthesized as inactive precursors- undergo proteolytic cleavage to become active

Caspase Activation Amplification Cascade

So How Are Initiator Caspases Activated to get the Process Going??

Main Pathways Regulating Caspase Activation During Apoptosis

Intrinsic Pathway- Mitochondrial MediatedMajor Pathway in Mammalian Cells – Outer Mitochondrial Membrane Permeabilization (MOMP)– Release of Cytochrome C from Mitochondrial Intermembrane

Space into Cytosol– Apoptosome Formation- Activation of Initiator Caspase – Effector Caspases Activated

Extrinsic Pathway- Signaling through Death Receptors– Ligand Bound Death Receptors– Adaptor Protein Association– Initiator Caspase Recruitment and Activation– Effector Caspases Activated

Intrinsic Pathway of Apoptosis Activation

Association of Adaptor with Procaspase allows

Procaspase self cleavage

MOMPs

cytochrome c Release

Apoptosome Formation: Adaptor (Apaf1), dATP

cytochrome c and procaspase complex

Active Initiator CaspaseCleaves Effector

Caspases Which now Cleave

Targets

Critical Regulators of Cell Death

Bcl-2 Family – Regulate whether MOMPs Occurs

Anti-Apoptotic Factors - Death Inhibitors

A) Function to Inhibit Cytochrome C release

Pro-Apoptotic Factors- Death Activators A) Bind and inhibit Death Inhibitors

B) Directly cause Permeabilization of Stimulate Release of Cytochrome C ( BAX AND BAK)

IAP Family (Inhibitor of Apoptosis)Bind Procaspases prevent activation

Bind Caspases and inhibit Activity

Survival Factor Signaling is Required to Prevent Apoptosis

Programmed Cell Death in Neuronal

Development

Survival Factors Signaling Keeps

Death Inhibitor Bcl-2 Active

No Survival SignalBcl-2 Complexes with BadCan’t preventBAK and BAX MediatedMOMPs

Extrinsic Pathway of Apoptosis Activation:Signaling through the Death Receptors

Ligand Bound Death Receptors

Adaptor Protein and Procaspase Recruitment

Initiator Caspase Activation

Effector Caspases Activated

Target cells :

Viral Infected Cells or Cancer Cells

Removal of Excess Lymphocytes afterInfection