Apoptosis Lecture

APOPTOSIS• Formed by Greek prefix apo- (from) and root ptosis (falling)• “leaves falling from a tree……”• the differentiation of fingers and toes in a developing human

embryo occurs because cells between the fingers apoptose; the result is that the digits are separate.

• Between 50 and 70 billion cells die each day due to apoptosis in the average human adult

• For an average child between the ages of 8 and 14, approximately 20 billion to 30 billion cells die a day

• Cells are born, live for a given period of time and then die-• Physiological cell death• Cell suicide• Cell deletion• Programmed cell death

AN APOPTOTIC CELL IN CULTURE

Collins JA, et al. 1997

THE APOPTOTIC PATHWAY

Triggers Modulators Effectors Substrates DEATH

. FADD

. TRADD

. FLIP

. Bcl-2 family

. Cytochrome c

. p53

. Mdm2

. Caspases . Many cellular proteins. DNA

. Growth factor Deprivation. Hypoxia. Loss of adhesion. Death receptors . Radiation . Chemotherapy

ApoptosisApoptosis is a cell death process which occurs during development and aging of animals.

Death by injuryThe cells swell and cell contents leak out, leading to inflammation of surrounding tissues

Death by suicideThe process is often called programmed cell death or PCD.

German scientist Carl Vogt was first to describe the principle of apoptosis in 1842.

In 1885, anatomist Walther Flemming delivered a more precise description of the process of programmed cell death.

The 2002 Nobel Prize in Medicine was awarded to Sydney Brenner, Horvitz and John E.

Sulston for their work regarding apoptosis

Etiology of cell death

Necrosis:• The sum of the morphologic changes that follow cell death in a living

tissue or organ

Apoptosis:• A physiological process that includes specific suicide signals leading

to cell death

Forms of cell death

"Classic"Necrosis Apoptosis Mitotic catastrophe

Passive Active Passive

Pathological Physiological or Pathological pathological

Swelling, lysis Condensation, Swelling, lysis cross-linking

Dissipates Phagocytosed Dissipates

Inflammation No inflammation Inflammation

Externally induced Internally or Internally induced externally induced

APOPTOSIS

In development, apoptosis removes unwanted cells; for example in separating the digits of the hand, culling excess neurons, eliminating inappropriately sensitive lymphocytes

In mature tissues that undergo renewal, e.g., blood cells, some epithelia, the outdated cells deliberately destroy themselves, if they canSome defensive cells die in the course of protecting the body, e.g. neutrophils, or in developing maximum affinity for attacking an invader, e.g. B lymphocytes

Apoptosis is used in running down unused or unwanted female reproductive tissue as it cycles, e.g., uterine endometrium, corpus luteum, breast gland epithelium

Most tumor cells grow; some become apoptotic

Infected cells may be removed by apoptosis

APOPTOSIS: Contexts II

In development, apoptosis removes unwanted cells, more examples: after matrix synthesis in the growth plate, in old bone awaiting resorption,

Apoptosis is part of running down unused or unwanted female reproductive tissue as it cycles, e.g., uterine endometrium, corpus luteum, breast secretory epithelium

In ovarian late atretic (degenerating) follicles, granulosa cells display hallmarks of apoptosis, e.g., fragmented nuclei, shrinkage, as they shed into the lumen

Some defensive cells die in the course of protecting the body: lymphocytes can kill each other, thus limiting immune reactions, & avoiding autoimmunity

APOPTOSIS: Contexts IIIIn these contexts, two kind of situation emerge: tidy coordinated loss (and sometimes replacement) of cells; and the messy battlefields of defense against infection, reaction to tumors, & wound repair

The macrophages go about their business of cell disposal very discreetly, without inflammation

This is not death with the headlights on and the traffic stopped; rather it is akin to the night-time anatomical grave-robbing of old, except that for apoptosis the body may be dismembered, but is not quite dead

APOPTOSIS: Contexts IV

Another context is all the other paths open to a cell, aside from apoptosis

Histology is first presented mostly as a described steady state - this is what the cells look like, and what they are doing as their normal function

However, the cells have many options, and are held by numerous controlling systems in this ‘steady state’

The options open to a cell can be categorized as: keep going/holding steady; advance; or retreat; as depicted on the next figures

Programmed cell death is the last resort of retreat

[Programmed cell death is not quite synonymous with apoptosis, since the latter term is restricted by some biologists to programmed cell death dependent on caspase enzymes]

senescence

transdifferentiate

APOPTOSIS: Contexts VI - Cell options

keep working

fuse

hypertrophy

enlarge & divide

apoptosis

stress responses

de-differentiate

shrink

RETREAT OR ADVANCE

APOPTOSIS: Contexts VII

This integrated control requires a host of chemical agents and factors, and has to take into account the many circumstances in which a cell finds itself

Few of these factors that regulate apoptosis: p53, Bcl-2 family, cytochrome c, TNF-, Fas ligand & receptor, caspases, endonucleases

However, the cells have many options, and are held by many controlling systems in this ‘steady state’

These categories - keep going; advance; or retreat - are mutually exclusive, so that control systems have to be interlocked, with inhibition of contrary activities, while stimulating a particular sequence and path

apoptosis

stress responses

de-differentiate

shrink

Apoptosis displays features of the cell states that often precede it

RETREAT OR

Apoptosis often occurs because the less drastic state of retreat cannot be maintained

membrane blebbing & changes

mitochondrial leakage

organelle

reduction

cell

shrinkage

nuclear fragmentation

chromatin condensation

APOPTOSIS: Morphology

membrane blebbing & changes


organelle reduction

cell shrinkage

nuclear fragmentationchromatin condensation

APOPTOSIS: Morphological events

organelle reduction

APOPTOSIS: Morphological events

sounds a rather coy term, but is intended to indicate that the organelles change and become non-functional, in ways often short of fast, outright destruction; e.g.,

Chiu R et al. A caspase cleavage fragment of p115 induces fragmentation of the Golgi apparatus and apoptosis. J Cell Biol 2002;159:637-648


organelle reduction

cell shrinkage

membrane blebbing

membrane changes


Minimum for cell death

The cell depends on the nucleus, so take this out of commission with enzymes for

Call in the macrophages with a surface marker

However, for orderliness, and to help the macrophages, other changes occur,

Wrapped - cell membrane integrity is kept

which convert the cell to wrapped, smaller, partially digested pieces

Bleb

Blebbing & Apoptotic bodies

The control retained over the cell membrane & cytoskeleton allows intact pieces of the cell to separate for recognition & phagocytosis by Ms

Apoptotic body

M M

Macrophage recognition of the apoptotic cell

Apoptotic body

M

M

membrane changes


Plasma membrane changes:

Phosphatidylserine is exposed externally

Membrane proteins lose their normally asymmetric distribution across the membrane

membrane changes, e.g.


organelle reduction

cell shrinkage


Time for some chemical agents

membrane blebbing

The cell depends on the nucleus, so take this out of commission with enzymes for


However, for orderliness, and to help the macrophages, other changes occur,

Cytochrome c

ENDONUCLEASES

CD14, & ligands for macrophageCD44

CASPASE proteases

CASPASE cysteinyl-aspartate-specific proteinase

APOPTOSIS: Signaling & execution

Tumor Necrosis Factor-

Apoptosis events

Apoptotic signalsFas ligand

Initiator caspases

Execution caspases

Cytochrome c

Apoptosis vs. Necrosis

• Necrosis– Swelling of cell– Disassembly of organelles– Membrane integrity

compromised– Release of cell contents– Often lead to inflammatory

response• Apoptosis

– Shrinkage of cell– Preservation of membrane

integrity– Chromatin condensation– Phagocytosed by other cells– No inflammatory response

Apoptosis and Programmed Cell Death (PCD)

• These two term are usually used interchangeably

• Apoptosis emphasizes more about the morphology (to be mentioned below)

• PCD emphasizes more about the orchestrated nature

• PCD is a broader term than apoptosis (PCD in plant, autophagic cell death, etc.)

So what is apoptosis?

• Regulated cell death

• Intrinsic mechanism

• Unique cell morphology

• Crucial in development and diseases

Morphology of Apoptosis

• Cell shrinkage (blebbing)

• Dark Nucleus (due to disintegration of chromatin)

• Apoptotic body

How Apoptosis was Discovered?

• First discovered in 1970’s during research of Caenorhabditis elegans development

• The discoverer, Dr. Horvitz, won 2002 Nobel Prize in Physiology or Medicine

• Has since been found in many higher animals (including human, of course).

Caenorhabditis elegans

• A nematode (app. 1mm long)

• A model animal in molecular and development biology research

• Genome determined

A perfect model for development biology

• Short life cycle (3 weeks)

• Existence of both allogamy and autogamy.

• Whole development is determined to the single cell level– Every C. elegans has exactly 1090 somatic cells– Each cell’s fate is invariant in all individuals

Apoptosis in C. elegans

• Of the 1090 cells C. elegans has, exactly 131 will undergo apoptosis, making the adult nematode has only 959 cells.

• Mutations affecting the death of these cells are identified. These mutants are viable

• ced-3 and ced-4 were first identified as pro-apoptotic genes (killer genes)

• ced-9 later was found to be anti-apoptotic genes

Not just in those worms!

• The gene product of ced-3 was found to be homologous to a mammalian protein ICE (later dubbed as caspase 1)

• Caspases were found in mammals, as well as other animals such as zebrafish and fruit fly

• Now apoptosis is generally believed to be a ubiquitous phenomenon among metazoa

Apoptosis in Development

• An efficient way to get rid of cells no longer needed

• Examples:– Digit development– Elongation of long bone– Amphibian metamorphosis

Apoptosis in Development

• Digit development• Elongation of long

bone• Amphibian

metamorphosis

STAGES OF CLASSIC APOPTOSIS

Healthy cell

DEATH SIGNAL (extrinsic or intrinsic)

Commitment to die (reversible)

EXECUTION (irreversible)

Dead cell (condensed, crosslinked)

ENGULFMENT (macrophages, neighboring cells)

DEGRADATION

STAGES OF CLASSIC APOPTOSIS

Genetically controlled: Caenorhabditis eleganssoil nematode (worm)

Healthy cell Dead cell Committed cell

ces2 ces1 ced9 ced3,4

BCL2 Caspases(proteases)

C. elegans genes == mammalian genes

Cells are balanced between life and death

DAMAGE Physiological death signals

DEATH SIGNAL

PROAPOPTOTICPROTEINS(dozens!)

ANTIAPOPTOTICPROTEINS(dozens!)

DEATH

APOPTOSIS: important in embryogenesis

Morphogenesis (eliminates excess cells):

Selection (eliminates non-functional cells):

APOPTOSIS: important in embryogenesis

Immunity (eliminates dangerous cells):

Self antigenrecognizing cell

Organ size (eliminates excess cells):

APOPTOSIS: important in adults

Tissue remodeling (eliminates cells no longer needed):

Virgin mammary gland Late pregnancy, lactation Involution(non-pregnant, non-lactating)

Apoptosis

Apoptosis

- Testosterone

Prostate gland


Tissue remodeling (eliminates cells no longer needed):

Resting lymphocytes + antigen (e.g. infection) - antigen (e.g. recovery)

Apoptosis

Steroid immunosuppressants: kill lymphocytes by apoptosis

Lymphocytes poised to die by apoptosis


Maintains organ size and function:

Apoptosis+ cell division

Cells lost by apoptosis are replaced by cell division

(remember limited replicative potential of normal cellsrestricts how many times this can occur before

tissue renewal declines)

X

APOPTOSIS: control

Receptor pathway (physiological):

Death receptors:(FAS, TNF-R, etc)

FAS ligand TNF

Deathdomains

Adaptor proteins

Pro-caspase 8 (inactive) Caspase 8 (active)

Pro-execution caspase (inactive)Execution caspase (active)

DeathMITOCHONDRIA

APOPTOSIS: control

Intrinsic pathway (damage):

Mitochondria

Cytochrome c release

Pro-caspase 9 cleavage

Pro-execution caspase (3) cleavage

Caspase (3) cleavage of cellular proteins,nuclease activation,

etc.

Death

BAXBAKBOKBCL-XsBADBIDB IKBIMNIP3BNIP3

BCL-2BCL-XLBCL-WMCL1BFL1DIVANR-13Several viral proteins

Apoptosis and Proliferation: Two faces of a coin

Diseases Related with Apoptosis

• Decreased rate of apoptosis let cells that usually die survive. Why is this bad?

• When cells have DNA damage beyond repair, they normally undergo apoptosis. If they somehow escape apoptosis (mostly related to p53 mutation), cancer might follow

• Some viruses can hijack the host transcriptional system to produce anti-apoptotic proteins to prevent their host from apoptosis

Diseases Related with Apoptosis

• Too much apoptosis causes atrophy

• For aging people, their neurons may undergo apoptosis, causing neurodegenerative diseases (Alzheimer's, Parkinson’s)

• Alcohol-induced liver diseases

• Myocyte degeneration

Caspases: Executioner of Apoptosis

• A family of Cysteine-aspartic-acid-proteinases (14 is known)

• With cysteine in active site• Cleave after aspartic acid• Normally in inactivated form (pro-enzyme)• Can be activated via cleavage after aspartic

acid (hint: self-activation)• Use positive feedback to form a cascade

Initiator and Effector Caspases

• Initiator caspases (2, 9, 8, 10): – Activated by intrinsic or extrinsic stimuli

– Cleave and activated effector caspases:

• Effector caspases (3, 6, 7): – Activated by initiator caspases

– Cleave and activate themselves and carry out caspase cascade

– Cleave many other proteins to fulfill the whole apoptosis phenomena

What lead to caspases?

• Two general pathways involved– Death-receptor– Mitochondrial

• Different initiator caspases are used

• Both pathways converge in caspase 3, the main effector caspase

Death-receptor Pathway

• Triggered by extrinsic ligand (“order of seppuku” ?)• A family of membrane receptors, generally called death-

receptors, regulates this pathway. e.g. TNF I, CD95• These receptors recruit procaspase-8 via an adaptor protein

(FADD)• Caspase-8 activation by proximity induction• Effector caspases…

Mitochondria Pathway

• Triggered by intrinsic signals, normally when the cell is damaged beyond repair

• Activation of some members of an important protein family (bcl-2 family)

• Mitochondria release cytochrome C• Formation of apoptosome (cytochrome C, Apaf-1, caspase-9)• Caspase-9 is activated

Early in apoptosis, mitochondria are triggered by multiple stimuli to release proteins that induce apoptosis. These include: oxidants, bax (a pro-apoptotic protein that targets mitochondrial membranes), Ca2+ overload, active caspases, and perhaps ceramide

The following caspase-activating proteins are then released from the intermembrane space:1. Cytochrome c (SMAC)2. AIF (apoptosis-inducing factor) OMNI3. And procaspases like procaspase-3 and caspase-2

Cytochrome c binds with Apaf-1, which then associates with procaspase- 9. This triggers caspase-9 activation. The complex of cytochrome c-Apaf- 1-caspase-9 then activates caspase-3 proteolytically.AIF also processes procaspase-3 to initiate caspase-3 activation.This cascade by caspases (cysteine proteases that cleave substrates at aspartic acid residues) culminates in apoptosis.

There are two general mechanisms:1. The outer mitochondrial membrane ruptures due to

expansion of the matrix space and organellar swelling.2. This releases cytochrome c, AIF, etc.How are cytochrome c and other caspase-activating pro

released from mitochondria?In this scenario, the mitochondrial inner membrane potential

drops, indicating the openings of channels known at permeability transition pores. These pores are composed of both inner and outer membrane proteins.

When the pores open, water and solutes enter the matrix, causing matrix swelling and outer membrane disruption.

2. The other mechanism also involves the opening of

channels. But, in contrast, these permeability transition

pores open only in the outer membrane and do not result in organellar swelling.

These transition pores allow cytochrome c and other

proteins to move from the intermembrance space into the

cytosol.

•Permeability of the membranes appear to be

enhanced by calcium, pro-oxidants, and several

apoptosis-related proteases (caspases)

•Bcl-2 and Bcl-2-like proteins increase resistance to

pore opening

Bcl-2 family• A protein family with its membranes related

tightly with apoptosis• At least 14 members• 3 Groups

Bcl-2 family

• Group 1 contains BH4 domain, usually anti-apoptotic

• Group 2 and 3 usually pro-apoptotic

• Yin and yang

• Cell survival/apoptosis depends on balance of these two anti and pro-apoptotic proteins

Possible Mechanism of Bcl-2 Family Protein Function

• The pro-apoptotic members of Bcl-2 family for a pore on mitochondrial membrane through which cytochrome C can be released

• They can also directly activate caspase via adaptor molecules

• They can modulate mitochondrial membrane to control the chemical and energy balance across the membrane

• The anti-apoptotic members bind to the pro-apoptotic members to form a heterodimer, so as to disrupt the aforementioned functions

Ways to detect Apoptosis

• Morphological evidence (mentioned above)

• Phosphatidylserine translocation

• Caspase activation

• DNA fragmentation

Phosphatidylserine Translocation

• Phosphatidylserine (PS) is a kind of phospholipid that constitutes the plasma membrane

• In normal cells, PS always resides in the cytosol side of the plasma membrane

• In apoptotic cells, PS quickly translocates to the exterior side of plasma membrane, which tags the apoptotic cells for engulfment by other cells (the “eat me” signal)

• The translocated PS can be detected by annexin-V binding

Phosphatidylserine Translocation (Detected by Flow Cytometry)

DNA Fragmentation

• In normal cells, DNA are wired around protein spindle called histones

• DNA and histones form units called nucleosomes

• In apoptotic cells, cleaved by DNase, nucleosomes are cut loose, like beads come off a string

DNA Fragmentation

• DNA of apoptotic cells subject to electrophoresis

• The DNA in nucleosomes cut loose have lower MW than intact DNA, thus move faster in electrophoresis

• Result in a “ladder” in the gel

CELL SURFACE DEATH RECEPTORS

Calbiochem, Inc

APOPTOTIC SIGNALING PATHWAYS

Fas

Procaspase 3

Caspase 8

Caspase 3

Bid

tBid

FADD

Procaspase 8

Bax/Bak

Procaspase 9

Caspase 9

Procaspase 3

Caspase 3

Cytochrome c

Growth Factor WithdrawalIrradiationLoss of Matrix ContactGlucocorticoids

?

Bcl-2Bcl-X

DNA Fragmentation

iCAD

CAD

Apaf-1

m

EXTRINSIC PATHWAY INTRINSIC PATHWAY

C-FLIP

IAP’sSmac

CASPASES

Caspase-1 (ICE)

Caspase-2 (ICH-1, Nedd-2)

Caspase-3 (CPP32, Apopain, Yama)

Caspase-4 (ICH-2, TX, ICEreıı)

Caspase-5 (ICErelııı, TY)

Caspase-6 (Mch2)

Caspase-7 (ICE-LAP3, Mch3, CMH-1)

Caspase-8 (FLICE, Mch5, MACH)

Caspace-9 (Mch6, ICE-LAP6)

Caspase-10 (Mch4)

SUBSTRATES for CASPASES

... PARP

... DNA-PK

... pRb

... Lamins

... NuMA

... Fodrin

... -Aktin

... Mdm2

... Cyclin A2

... Presenilin

... Others

WHERE can APOPTOSIS be WHERE can APOPTOSIS be ENCOUNTEREDENCOUNTERED ? ?

... Growth of Embrio

... Tissue Homeostasis

... Immunology

... Chronic viral diseases

... Neurodegenerative diseases

... Reperfusion injury

... Insuline-dependent Diabetes

... Atheroschlerosis

... Miyokard Infarction

... AIDS

... Development and Treatment of Malignancies

Significance of apoptosis in cancer1) Tumor induction• Apoptosis is an important regulator of

homeostasis--elimination of cells with damaged genome or growth in marginal (hypoxia)

• Apoptosis ensures the integrity of tissues

2) Viral transformation (adenoviral model) uses both proliferation stimulus (E1A) and anti-apoptotic (E1B) mechanisms.

3) Anti-neoplastic agents can cause an apoptotic tumor cell death

Moduators of Apoptosis--bcl-2 Family

• Bcl (B cell lymphoma)-2 cloned from chromosomal translocation

• Protect from death due to growth factor withdrawal

• functional homologue of ced-9

• Pro- and anti-apoptotic members

Bcl-2 Family

BH4 BH3 BH1 BH2 MA

Dimerization

Pore Formation

Bcl-2Bcl-xL

MABH4 BH1 BH2CED-9

BH3 BH1 BH2 MABax

BH3 MA

Anti-apoptosis

Pro-apoptosis

Bik

BH3BadBidEGL-1

Effectors of Apoptosis--Caspases

• Proteases synthesized as proenzyme (initiator vs effector)

• Cysteine in active site• Aspartate key to

recognition sequence (iCAD)

• ced-3 prototypical member

CASPASES

Pro(DD’s)

Large( 20 kd)

Small( 10 kd)

DX DX

Active Caspase

Caspase Substrates Category Substrate Caspase Effect Proposed Role in Apoptosis

Structural Nuclear Lamins 6 Degraded Disassembly of nuclear matrix

-Spectrin 3 Degraded Disassemble cytoskeletal-membranecontacts

FAK 7,6 Inactivated Disassemble focal adhesions

Gelsolin 3 Activated Disassemble actin filaments, blebbing,DNA cleavage

Actin 3,1 Degraded Disassemble actin filaments

-catenin 3 Degraded Disassemble cell-cell contacts

NuMA 3,4,6,7 Degraded Disassemble chromatin/matrix contacts

Vimentin 3 Degraded Disassemble intermediate filaments

Signaling MEKK1 3 Activated Activate SAPK

Stat1 ? Inactivated Inhibit IFN signaling

PKC , 3 Activated Nuclear condensation/fragmentation

D4-GDI 1,3 Inactivated Deregulate Rho GTPase

Akt-1 ? Inactivated Inhibit survival pathway

NF-B p50, 65 3 Inactivated Inhibit survival gene induction

Cell cycle DNA replicationcomplex C

3 Inactivated Inhibit DNA replication

mdm2 3 Altered Inhibit p53

Rb 3 Inactivated Release E2F-1

DNA repair PARP 3 Inactivated DNA nicks not recognized

DNA-PK 3 Inactivated Inhibit repair of strand breaks

Other IL-1 1 Activated Inflammation

IL-16 3 Activated Inflammation

IL-18 1 Activated Inflammation, induce IFN-

ICAD/DFF-45 3 Inactivated Allow translocation of CAD intonucleus, DNA fragmentation

Bcl-2, Bcl-XL 3 Altered Functions like BAX

Bid 8 Activated Translocates to mitochondria

Nedd4 1,3,6,7 Regulate ubiquitination

Inhibitors of Apoptosis--IAP’s

• Bind procaspases to prevent activation– initial description of bacculovirus product (p35)– contain metal-binding (~80aa) BIR repeats– number and sequence of BIR’s determine specificity of procaspase inhibition– many contain RING fingers

• Survivin– expressed in many fetal tissues– expressed in many colon cancers

• IAP modulators– inhibitors of inhibitors– Smac/Diablo

Pathways of Apoptosis--Mitochondria

• Cytochrome c released

• Cytochrome c binds Apaf-1 (ced-4 homologue)

• Apaf-1/cytochrome c complex binds caspase 9 (ced-3 homologue) and causes autoactivation

DEATH

CASPASE 3OTHERS

MITOCHONDRIA

CASPASE 9

TNF Receptor Family Signaling

Fas

TNFR1 TNFR2

TRADD

Procaspase 3

Caspase 8

Caspase 3

TRAF2

FADD

Procaspase 8 TRAF1

NFkB Activation andGene Induction (eg. SOD, IAP’s)

Pro-Apoptotic

Anti-Apoptotic

Anoikis--Death by loss of matrix contact

Adhesion--ILK, FAK, Shc

PI-3K

AktBad, Bim, ProC9

ApotosisGSK-3

CyclinD1

G1S

IGF-1

Rb-E2F

PTEN

Based on Yu, et.al. Mol. Cell. Biol.21:3325.

p53-Mediated Apoptosis

H2N COOH

TA PP DB T CTD

TA Transactivation domain recruits proteins involved in transcription--eg TATA box binding proteins and associated factors--also binding site for oncogene Mdm2 which targets p53 for ubiquitination (p19ARF inhibits)

PP Polyproline domain critical for the induction of apoptosis and transcriptional repression, but not activation

DB Sequence-specific DNA binding domain region of most mutations in tumors

T Tetramerization domain important for oligomerization and DNA binding

CTD COOH-termainal domain that is a negative regulator of the transactivating domain

Cell cycle and apoptosis

• p53 bax and bcl-2• overexpression of

E2F1 causes apoptosis

• p21 binds caspase 3 and is a substrate for caspase 3

M

G1

G0

S

G2

Rb

Rb

E2F1

E2F1

Cyclin D

Cyclin Ecdk4/6

cdk2Cyclin A

cdk2

Cyclin A

cdc2

Cyclin B

cdc2

PP

PP

p21

p27

p15

p16

p53 TGF

p53

bax

Adapted from Lundberg and Weinberg Eur. J. Cancer 35:531

R

E2F1

Inactive

Anti-neoplastic drugs and apoptosis

• Caspase 9 or Apaf-1 deficient cells are resistant to many forms of anti-neoplastic therapy (irradiation, etoposide, doxorubicin, cis-platin)

– These agents work primarily through p53, but there are poorly understood p53-independent pathways as well

– Silencing mutations in p53 are one mechanism of drug resistance– p53 also appears to be the target of numerous oncogenes (E1A, c-myc)

and p53 loss replaces E1B function in adenoviral transformation• 5’-Fluoruracil appears to work through the Fas pathway in some tumors

– p53 and reactive oxygen can induce FasL and Fas expression– Fas pathway is minor in most tumors, but neuroblastomas have silenced

the caspase 8 gene– FADD or caspase 8 deficient fibroblasts are resistant to FasL and TNF,

but sensitive to doxorubicin and etoposide• Akt pathway is important in tumor survival

– PTEN inactivation is correlated with poor prognosis in a number of tumor types

Induction of apoptosis in tumors by cytolytic immune effectors

• Immune cells can attack tumors through the extrinsic pathway

– TNF– FasL

• The granule-exocytosis pathway is more important in tumor control

– CD8 effectors– Natural Killer (NK) Cells– Granzymes

• Serine proteases stored in intracellular granules

• Granzyme B is an aspase (not a caspase) that can bypass initiator caspases to activate effector caspases or their substrates (eg. iCAD)

• Other granzymes with different specificities--substrates unknown

• Entry into target cells requires perforin

APOPTOTIC SIGNALING PATHWAYS

Fas

Procaspase 3

Caspase 8

Caspase 3

Bid

tBid

FADD

Procaspase 8

Bax/Bak

Procaspase 9

Caspase 9

Procaspase 3

Caspase 3

Cytochrome c

Growth Factor WithdrawalIrradiationLoss of Matrix ContactGlucocorticoids

?

Bcl-2Bcl-X

DNA Fragmentation

iCAD

CAD

Apaf-1

m

EXTRINSIC PATHWAY INTRINSIC PATHWAY

Granzyme B

APOPTOSIS: control

Physiological Intrinsicreceptor pathway damage pathway

MITOCHONDRIAL SIGNALS

Caspase cleavage cascade

Orderly cleavage of proteins and DNA

CROSSLINKING OF CELL CORPSES; ENGULFMENT(no inflammation)

APOPTOSIS: Role in Disease

TOO MUCH: Tissue atrophy

TOO LITTLE: Hyperplasia

NeurodegenerationThin skin

etc

CancerAthersclerosis

etc

APOPTOSIS: Role in DiseaseNeurodegeneration

Neurons are post-mitotic (cannot replace themselves;neuronal stem cell replacement is inefficient)

Neuronal death caused by loss of proper connections,loss of proper growth factors (e.g. NGF), and/or

damage (especially oxidative damage)

Neuronal dysfunction or damage results in loss of synapses or loss of cell bodies

(synaptosis, can be reversible; apopsosis, irreversible)

PARKINSON'S DISEASEALZHEIMER'S DISEASE

HUNTINGTON'S DISEASE etc.

APOPTOSIS: Role in DiseaseCancer

Apoptosis eliminates damaged cells(damage => mutations => cancer

Tumor suppressor p53 controls senescenceand apoptosis responses to damage

Most cancer cells are defective in apoptotic response(damaged, mutant cells survive)

High levels of anti-apoptotic proteinsor

Low levels of pro-apoptotic proteins===> CANCER

APOPTOSIS: Role in DiseaseAGING

Aging --> both too much and too little apoptosis(evidence for both)

Too much (accumulated oxidative damage?)---> tissue degeneration

Too little (defective sensors, signals?---> dysfunctional cells accumulatehyperplasia (precancerous lesions)

OPTIMAL FUNCTION (HEALTH)

APOPTOSIS

APOPTOSIS

AGING

Neurodegeneration, cancer, …..

Summary

• Apoptosis is a highly-regulated process that results in an efficient removal unwanted cells. It is greatly involved in development and prevention of oncogenesis

• Apoptosis is a complex process that involves interactions between many factors. The core components of apoptosis are the caspases

• Apoptosis is very important for homeostasis of the organism. Abnormal apoptotic rate may cause severe diseases

Apoptosis Lecture

Documents

Transcript of Apoptosis Lecture