Cell Death It is estimated that 10 10 cells are born and die each day in a human body (10 14 ). Apoptosis: Greek word meaning “ a falling away”, proteolytic.

Cell DeathIt is estimated that 1010 cells are born and die each day in a human body (1014).

Apoptosis: Greek word meaning “ a falling away”, proteolytic cascade leading to DNA fragmentation, membrane blebbing, and phagocytosis of cell.

Necrosis: Greek word meaning “to make dead”. Membrane pumps fall, causing cell to swell, burst and releasing digestive enzymes into ECM

Apoptosis is essential for normal growth and development, tissue homeostasis, as well as defense against pathogens.

Decreased apoptosis can lead to cancer, autoimmunity, and viral infections. Excessive Apoptosis can lead to AIDS, Neurodegenerative disorders, and Stroke.

Ultrastructural Features of Apoptosis

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Cell Lineage in the C. elegans embryo

Nature Structural Biology 8, 394 - 401 (2001)

Conserved Apoptotic Pathway

The Caspase Family

Science. 1998 Aug 28;281(5381):1309-12

The Integrated Apoptotic Pathways

TNFRI

Fas

Dr3

Dr4

Dr5

TNFR IICD40CD30CD27LTR4-1BBOX40NGFRHVEMGITRRANK

PV-T2

PV-A53R

TRID

OPG

DEATHDOMAIN

The Tumor Necrosis Factor Receptor Family

Dr6

Type IType II

DeathDomains

TRAF Proteins

Fas

Dr4

Dr5

TNF-R1Dr3

Dr6

DcR1

DcR2

Apoptosis

Immune & InflammatoryResponses

FADD

Caspase-8

RIP

TRADD

TRAF2Death EffectorDomains

Caspase 3

Caspase 7 Caspase 6

Survival signals

FADD

{{

mitochondr

ia

RelB

c-RelRelA (p65)

NF- B1p105 (p50)NF- B2

p100 (p52)IB-

Bcl-3

IB-IB-

DNA-BindingDimerization

NLS

Rel - Ankyrin Family

IB-

LPS

TNF-

IL-1

IL-2PHACalcium ionophoreAnti-CD28Anti-CD3Anti- TCRTCR-specific antigen

monocyteRat-2 fibroblastfibrosarcomahepatoma (Hep G2)T cell lines

T cell linesT-cell linesT-cell linesT-cell linesT-cell linesT-cell linesT-cell lines

protein synthesisinhibitors

phorbol esters

Anti IgM

HTLV I tax

ds RNA

AdenovirusCMVSendaiHerpes simplex 1Human herpes 6

Inducers of NF- BInducer Cell-Type Inducer Cell-Type

hepatoma (Hep G2)

pre-Bsplenic Bfibrosarcomamonocytemacrophage

pre-B (70Z/3)splenic B

splenic B

plasmacytomaT cell lines

osteosarcomaHeLafibrosarcomafibroblastsUV lightH 22

Okadaic Acidc-AMPTheilera parva

pre-BT cell linesHeLasplenic B

O

TIMDomains

ASK1

JNK

MKK4/7

IkB

p65 p105

IKKIKKIKK

NIK?

p100

TRAF Proteins

Immune and Inflammatory Responses

p65 p50 p52 RelB fosJun

?

1 2 3 4 5 TRAF-CZinc Fingers

TRAF-NN C

RingFinger

DeathDomains

??

p38 PI3KERK1/2

TRADD

RIP

type 1 type 2GCK/GCKR

Surface Receptors

Ig light chainIL-2R chainTCR chainMHC class I (H-2K )invariant chain Ii-2 microglobulinMHC class II (E, E)

IL-2IL-6IL-8GM-CSFG-CSFTNF-(cachectin)TNF- (lymphotoxin)-interferonproenkephalin

Acute Phase

angiotensincomplement factor Bcomplement C4Serum amyloid A

c-myc

HIV

SV40

cytomegalovirus

adenovirus

GGGACTTTCCGGGAATCTCCGGGAGATTCCGGGGATTCCCGGGAATTTCCGGGACTTTCCGGGACTTCCC

GGGATTTCACGGGATTTTCCGGAATTTCCTGGGAACTACCGGGGAATCTC severalGGGGCTTCCCGGGAAATTCCGGGACGTCCC

GGGATTTCCCGGGAATCCCCGGGAAGTCCCGGGACTTTCC

GGGAAAACCC

GGGACTTTCCGGGACTTTCCGGGACTTTCCGGGAAGTACCGGGACTTTCCGGGGATTTCCGGGACTTTCC

NF-B regulates a broad range of genes.GGGRNNYYCC

b

Bcl-x GGGACTGCCCBfl-1 GGGGATTTACCytokines

Viruses

Apoptosis

TNF- Tumor Deathin some cancers

TNF-Actinomycin D

Enhanced Death

TNF-NF-B Deficient Enhanced Death

NF-B-Regulated Genes InhibitTNF--Mediated Cell Death

TNF--Mediated Upregulation of Bcl-xand Bfl-1 Requires NF-B Activity

0 15 30 0 15 30

TNF- (min)

A549 42-7-8 Bcl-x

Bfl-1

BAG-1

GAPDH

0 2 4 0 2 4

TNF- (hrs)

A549 42-7-8

The Bcl-x NF-B Cis Element is Capable of RelA Mediated Transcriptional Activation

luciferase

NF-B Cis Element

Bcl-x WT Promoter

luciferaseBcl-x Promoter B-

0

5

10

15

20

25

30

Fo

ld A

ctiv

atio

n

WT Promoter B- Promoter

p65 - + - +

Establishing 42-7-8 Cell Lines ExpressingBcl-x or Bfl-1 at Physiological Levels

Bcl-xProtein

Tubulin

- + - + - +

TNF- Stimulation

A549

42-7

-8

Bcl-

x

Bfl-1Transcript

GAPDH

- + - + -TNF- Stimulation

A549

42-7

-8

Bfl

-1

.

.

100

80

60

40

20

0

% o

f C

on

trol

A549 42-7-8 Bfl-1Bcl-x

MTT ASSAY

IB*-Expressing Cells are Sensitized toTNF--Mediated Cell Death, which is

Inhibited by Expression of Bcl-x or Bfl-1

IB* ExogenousBcl-x

CD40 Survival Signaling through NF-B-Mediated Upregulation of Bcl-x and Bfl-1

FasCaspase Activity

Apoptosis

CD40 NF-BBcl-xBfl-1 Survival

Chemotherapy Drugs Differentially Upregulate the Expressionof Bcl-x and/or Bfl-1 through an NF-B-dependent Pathway

A549

42-7-8

Adriamycin

VP-16

0 10 20 30 40 M

M0 100 200 300 400

CDDP

0 2.5 5 7.5 10 g/ml

120

100

80

60

40

20

0

100

80

60

40

20

0

100

80

60

40

20

0

A549 Cells Are Sensitized to Chemotherapy-MediatedApoptosis Upon Inhibition of NF-B Signaling

Perc

en

tag

e o

f C

ell

Su

rviv

al

The Enhancement of Chemotherapy-Induced CellDeath Through Manipulation of Signaling Pathways

Chemotherapy

Pro-apoptotic signal

Anti-apoptotic signal1. NF-B-Mediated Upregulationof Bcl-2 Family Members2. Other pathways (AP-1?)

Death

Chemotherapy

Pro-apoptotic signal

Anti-apoptotic signal1. NF-B-Mediated Upregulationof Bcl-2 Family Members2. Other pathways (AP-1?)

EnhancedDeath

JNK Activation

DeathDomains

FADD TRADD

RIPFADD

Caspase 8/10

TRAF Proteins

Apoptosis

NF-B ActivationType I

FLIP

IAPs

Bcl-x/A1

Bcl-2/Bcl-XcIAP-1/c-IAP-2FLIP

TIMDomains

Ubiquitination

Caspase 3/6/7

Type II

Activation &Differentiation