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(, ) 30%
40%
30%

SOP
12 (2~3)


Apoptosis Disease
()


Cell fate in multicellular organism
DIFFERENTIATION

PROLIFERATION

Cell fate decided by survival


and promoting signals
Survival factors: for cell
maintenance
Promoting factors: for cell
proliferation, differentiation,
apoptosis and transformation
APOPTOSIS

TRANSFORMATION

PROLIFERATION
DIFFERENTIATION

APOPTOSIS

Cell Death

Majno, G. and I. Joris, Apoptosis, oncosis, and


necrosis. An overview of cell death. Am J Pathol,
1995. 146(1): p. 3-15.

(Accidental cell death)


(Apoptosis)

dye
exclusion

trypan blue
DNA

(Necrosis)
(Necrobiosis)

150Rudolph
Virchow (1902-1982)
(Necrosis)
(Necrobiosis)
(Necrosis)

(Necrobiosis)

(Physiological cell
death)

(Death by murder)(Death
by suicide)

(Accidental cell death)


(Necrosis)
(Cell suicide)
(Physiological cell
death) (Programmed cell
death) (Apoptosis)

(Accidental
cell death) (Necrosis)

(Ischemic cell death)

Guido MajnoIsabella Joris


(Oncosis)
(Coagulation necrosis)
ATP

(Blebbing)
(Budding)

(necrosis)

(necrosis)

necrosisapoptosis

Necrosis: The death of living cells or

tissues. Necrosis can be due, for example,


to ischemia (lack of blood flow). From the
Greek "nekros" (dead body).

Concept of the Apoptosis


Programmed cell death: a form of
cell death in normal embryogenesis
(Glucksmann, 1950s)
Shrinkage necrosis: a distinct
mode of cellular death (Kerr, 1962)
-Apoptotic bodies (arrowed) in liver
lobe atrophy after portal vein ligation

Apoptosis: distinct mechanism


of controlled cell deletion that is
complementary but opposite to
mitosis (Kerr, Wyllie, Currie, 1970)

apoptosis
apoptosisapo
ptosisApoptosis

1972Kerr
apoptosis

programmed cell
death ,PCD

Apoptosis
is a highly organized orchestrated form of cell death
(cellular suicide; programmed cell death)
Important role in development and homeostasis; embryo
development, synaptogenesis, tissue turnover, immune
response
Apoptosis malfunction: too little apoptosis - cancer and
autoimmune disease; too much apoptosis neurodegeneration (Alzheimers)
Apoptosis can be triggered by stress - oxidants (ROS), DNA
damage agents (uv, irradiation), chemotherapeutic drugs

Morphological assessment
control

Light microscopy:
- DNA condensation, nuclear
fragmentation, apoptotic bodies

Electron microscopy:
- Scanning EM
- Transmission EM

apoptotic

Apoptosis is a highly organized


orchestrated form of cell death

Paradox:
Life is dependent on cell death

Demonstration of single HT-29/B6 apoptotic cells.


1.
2.

Apoptosis Function

Reduce energy lost


Prevent cancer development
PCD
Homeostasis

Apoptosis is all around us

Apoptosis Inducer

infected with a virus


undergoing stress conditions (such as
starvation)
ionizing radiation
toxic chemicals

In plants as well

In plants as well (2)

Transduction ducts ( )

Necrosis vs. Apoptosis

Apoptosis programmed cell death


Necrosis un-programmed cell death

Necrosis vs Apoptosis
Chromatin compaction/margination

swollen cells & disrupted organelles


shrunken masses of cytoplasm with Nuclear fragmentation; apoptotic bodie
Phagocytosis: epithelial cells, phagocyte
intact cellular organelles

Apoptosis progression

,
,

DNA ,

1.

2.

buding


apoptosis

DNAHoechst 33342
Hoechst 33258, DAPI
DNA
DNAA-T

HoechstDNA
1mg/mlPBS
2~5mg/ml
DAPI
1mg/ml
0.5 ~1mg/ml

rippled
creased
a
b

Execution phase

DNA

pro-apoptosis nuclei

cavitations
a

apoptosis

TEM

SEM


1.

DNA Ladder

DNA

DNase

DNA Ladder
DNA2001
DNA
180200bpDNA

DNA fragmentation: biochemical hallmark

DNA cleaved into non-random fragments


Targets of endonuclease attack: linker regions between nucleosome
180-200 bp fragments & multiples of this unit
Agarose gel
electrophoresis

Chromatin

Fragmented
Chromatin

,
DNA,
50~300kbpDNA, 180~200bp

DNA
(DNA ladder)
DNA

DNA ladder
DNA32PATPTdTDNA

DNA ladder

DNA

,50~300kbp
DNA
50~300kbpDNA

DNA

DNA
DNA
DNA

DNA Ladder

1x1071%SDSRnaseA5mg/ml
56C 2h K2.5mg/ml37C2h
13000rpm5min
1/103M2.5DNA4C

14000rpm15min
TE bufferDNA Loading Buffer 1.2%
EB
Brown,D.G., Sun, X.M., and Cohen, G.M.(1993)
Dexamethasone-induced apoptosis involves cleavage of
DNA to large fragments prior to internucleosomal
fragmentation. J. Biol.Chem. 268, 3037-3039

Multiples of 180~200 bp
fragments

Ladder


DNA

70%in PBS4C
PBS1000rpm 10min
RNase A0.5mg/ml37C30min
PI50mg/ml15min
DNA

Fluorescence Detectors
Laser

Freq

FALS Sensor

Fluorescence

Fluorescence detector
(PMT3, PMT4 etc.)
Purdue University Cytometry Laboratories

FSCSSC
FSCSSC

DNA
1

2 DNA(DI)DNA
3

Sub-G0/G1 peaks --- PI staining

Comet assay

TUNELTerminal deoxynucleotidyl transferasemediated dUTP nick-end-labeling

, DNA
3-OH
TdT

DNA3-

DNA
3-OH


terminal -deoxynucleotidyl transferase mediated nick end
labeling, TUNEL

TUNEL

TUNEL assay

2.
(caspases)
Caspases8910
Caspases367912

Caspase-3

Caspase
caspase-3
Caspase-332KD
Caspase-3
17KD12KD

Western blot Procaspase-3


Caspase-3ADP- [polyADPribosepolymerasePARP] apoptosis

Caspase-3western
blot

PBS
SDS-PAGEPVDF
5%blocking1.5~2h4C
anti-Caspase-3 antibody 1~2h4C
TBS-T0.05% Tween 20TBS3
5~10min/
HRP-1~2h TBS-T3
5~10min/
ECL

2
Caspase-3DEVD-XD-X
Ac-DEVD-AFC
AFCAFCAFC
AFCcaspase-3
Caspase-3

1. PBS

2.

3. Ac-DEVD-AFCcaspase-337C1h
4. Polarstar380nm
430-460nm

PS
:

PS

Annexin-V
PS

DNA


Annexin-V

Phosphatidylserine, PS
PS

Annexin-V35~36KDCa2+
PS
Annexin-VFITCPEbiotin
Annexin-V

(propidine iodide, PI)


PI
Annexin-VPI

Annexin V Assay

Annexin-V

1. PBS2
0.5~1106
0.25% PBS
2. 100ul Binding BufferFITCAnnexin-V20ug/ml
10ul30minPI50ug/ml5ul
5min400ul Binding Buffer
FACScan
1h
3. AnnexinV-FITCPI

1.
2.

Annexin V/PI
Annexin V-/PI-
Annexin V+/PI+
Annexin V+/PI-

AnnexinV-FITC PSPS
apoptosis

Annexin V Assay

DYmt

DNA
DYmt

Rhodamine 123DiOC6JC-1TMRM

Rhodamine 1231mM
DiOC625nMJC-11mMTMRM
100nM37C30min

1 pHpH

apoptosis

apoptosis
C
(cytochrome C)
apoptosis
membrane potential
JC-1 Mitochondrial
Potential Sensors from red (~590
nm) to green (~525 nm)

apoptosis

1.
2.

3.DNase caspases
4.


cAMP
+
Ca2+

DNase
Caspases

Death factors
Bind to death
receptors[cluster and
trimerise]
Recruits FADD
DED

Recruits pro-caspase 8
[induced proximity]

E.g.: FasL/TNF

E.g.: Fas/TNFR

cAMP
Ca2+

DNase
Caspases

PT pore
PT pore cyclosporin A SH bongkrek acid
PT pore
PT pore
AIF
C


1. PT

2. bcl-2orBax

3. bcl-2m; Bax
m

bcl-2 (ced9)
P35:CaspasesP35 Caspase

Caspases
CrmA

P35CrmA

bcl-2

ced-9

1-4bcl-2


()
1.DNAPARPP53
NADATP

2.

2+/Mg2+
3.Ca
NF-kB
2+
4.Ca

2.

DNA

Caspases
Dnase

2 main integration pathways

Death receptor pathway


Mitochondrial pathway

Both pathways converge


to a common execution
phase

(caspases)
(1)

(2)Bcl-2
(3)

Caspase family

1C
QACR/QG
2290004900029-49KD
P20P10

P20/P10

Caspase
inititaoreffector

Caspase

caspaseN-
N-

Caspase

Caspasecaspaseinitiator
caspasecaspase-8,-10,-9
Caspase

capases
Caspase
Caspase

hetero-activationcaspase
caspase
Caspasecaspase(executioner
caspase)Caspase-3,-6,-7Caspase
Caspase
Caspase

Apoptosis common execution phase

FLIPs : Fas/TNFR1
DEDFADDCaspase-8
10
Caspase810

PT pore ( PT
PT pore


1.
(1)

HIVCD4+T

(2)
IL-2
ACTH
Zn2+

1
2

3.
1Bcl-2EIB IAP
2p53ICEFasBax
3c-myc

Bcl-2(B-cell lymphoma/leukemia -2)

*
*
*
*
*

2p53ICEFasBax
p5353kD p53

DNA+P53

DNA

DNA

P53

3C-myc

apoptosis

1.
2.

3.


Bcl-2---
p53

T
TB
TCR
T
negative selection

1.

P53

Bcl-2

Inhibition of apoptosis: Cancer

Deletion/mutation of
p53
express high levels of
Bcl-2
FAS signaling

2.

SLE1

Fasp53

IR


Excessive apoptosis:

Alzheimer disease

Loss of neural cells


Induced by b-amyloid peptides
b-amyloid peptides trigger toxicity via activation of caspase-8
and 9


Alzheimer disease, AD; Parkinson disease ;

AD
-
AD

Ca2+

VitE

[Ca2+]

HIVCD4+
1HIVGP120CD4+
2 (syncytia)
3Fas
4T
5MTNF
6tatCD4+OFRFas
7CD4+
CD4+

CD4
CD4

(
)
()

1.
2.

AIDS : HIVCD4+
(apoptosos)CD4+
CD4+HIV

Inappropriate apoptosis: HIV

Death of CD4+ T cells most by apoptosis


Expression of Nef gene in effected CD4+ T
cells

1. :

2. : NGF