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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