Methods for Apoptotic Assay

Apoptosis

A physiological mechanism to eliminate excess,

damaged or dangerous cells from an organism

without damaging surrounding cells and tissues

Necessary for normal embryogenesis

Maintenance of tissue homeostasis

Apoptotic morphology

Membrane blebbing

Aggregation of chromatin at the nuclear membrane

Ends with fragmentation of cell into small bodies

Begin with shrinking of cytoplasm and condensation

of nucleus

Formation of apoptotic bodies

Mitochondria become leaky due to pore formation

involving proteins of the bcl-2 family

Apoptotic Pathway

Death receptors

Membrane alteration

Protease cascade

Mitochondrial changes

DNA fragmentation

INTRINSIC PATHWAY Extrinsic pathway

Caspase3

caspase7

Apoptosis

procaspase9

cytochromeC

APAF-1 Apoptosome

XIAP

caspase9 caspase8

Procaspse3

procaspase7

Procaspase 8

Death receptors

Death inducing signaling complex

Ad

ap

tor

pro

tein

s

Death domain adaptor protein

TNF FASL Apo2/Trial

Death effector domain

The extrinsic death receptor pathway

Death receptors

The intrinsic apoptotic pathway

Caspase activation

Apoptotic insult

apoptosome

Caspase dependent cell death

AIF

chromatin

nuclease

chromatin

nuclease

DNA fragmentation

Protease cascade

*Conserved domains for caspase interaction:

Death Domain( DD) QACRG

Death Effector Domain( DED)– hydrophobic interaction

Caspase recruitment Domain( CARD)—electrostatic

interaction

* Amplify a suicide signal in the cell whose death pathways

have only weakly initiated

Caspase family

Death Effector domain

Caspase recruitment Domain

EGL1

CED-9

CED-4

CED-3

apoptosis

Nematodes

mitochondria

Cell death

CED-9

Pro CED-3

CED-4

Active CED-3

βNAC

Icd-1( inhibitor of cell death -1))

Early embryo arrest

Accumulation of extra die cells

Extra die cells in nematode show normal survival

BID, BIM

BCL-2

SMAC/DIABLO

IAP

APAF-1

caspase9

Caspase3,7

Cytochrome C/dATP

AIF

HTR2/OMI

EndoG

Apoptosis

Apoptotic stimulimammals

Reaper

Hid

Grim

Sickle

DIap1

Apaf1

Dronc

Drice

Cellular target

Apoptosis

Fruitfly

Eat me signal

PS ( phosphotidylserine) displayed on the plasma membrane on the dying cells

Engulfment receptors in mammals:

CD91, CD14, CD36, αvβintegrin, phosphotidyl serine receptor( PSR)

Cell Engulfment

ced1: engulfment receptor

ced-6: homologue to the mammalian PTB domain bearing

adaptor GULP

ced-2( CRKII)

ced5 ( DOCJ-180)

ced10( small GTP-ase Rac-1)

Cytoskeletal remodeling

Apoptotic cells

Engulfment of apoptotic cells

Phagocytic cells

DNA degradation

Phosphotidylserine

Cytoskeleton remodelingELMORAC1

Dock180

CED-5

CED-2CRKII

PSR

CD19

CD91

CD14

αvβintegrin,

αvβintegrin,

Scavenger receptor

CED-1CED-6 CED-7

Membrane blebbing

nematodes mammalians

Phosphotidylserine

Decision to die,

Execution of death, caspase activation

Engulfment signal, LPC….

degradation

Apoptotic induction, caspase activation

Eat me signal,CD31/ PCAM-1

psp

s

Anexin I

Anexin I

180 b.p.

Increased low molecular weight DNA in apoptotic cells

Terminal dideoxynucleotidyl transferase

annexinV: Ca+2 dependent phospholipid binding protein, high affinity to PS

Substrate cleavage

Caspase activation

Apoptin 誘發細胞凋亡機制之探討

Mechanisms Involved in Apoptosis

induced by Apoptin Associated Proteins

Chicken Anemia Virus

diameter 23 - 25 nm

single-stranded circular DNA (2319 nt)

polycistronic mRNA

VP1 51.6 kDa

VP2 24 kDa

VP3 13.6 kDa (Apoptin)

CAV infection cause depletion of erythroblastoid and lymphoid cell in young chicken

(Jerissen & Noteborn et al, J Viorogy,1992)

Apoptosis is responsible for the thymocyte depletion in young chicken

(Jerissen et al, J Virology,1992)

VP3 protein of CAV is sufficient to induce apoptosis in chicken mononuclear cells

( Zhung & Noteborn et al, Leukaemia1995)

Noteborn et al, Cancer Res, Carcinogenesis,1995

p53 Apoptin

Apoptosis

Bcl-2

BAG-1

CrmA

ICE

Apoptin induces apoptosis in human transformed and malignant cells but not in normal cells

Danen-Van Oorschot et al, PNAS 1997

Apoptin activity and localization in human normal vs, transformed/tumorgenic cells

Human cells Apoptin

Apoptotic activity localization

Normal primary None cytoplasmic

Tumor-derived/transformed yes nuclear

Noteborn etal, 1998, Mutation Research

The effect of transforming genes and UV-irradiation on Apoptin induced apoptosis

Donors of fibroblast Apoptin induced Apoptosis

Apoptin expression plus

none SV40 largeT UV

Healthy no yes no

Cancer prone no yes yes

Noteborn etal, 1998, Mutation Research

Apoptin –expressing adenoviral vector specifically kills tumor cells of xenogenic HepG2 tumor cells in Balb/Cnu/nu

Pietersen et al 1999 Gene Therapy

Apoptin induced apoptosis requires activation of caspases in human tumor cells

Oorschot et al 2000 J Virology

A tumor specific kinase activity regulate the viral death protein Apoptin

Rohn et al 2002 JBC

Apoptin induces tumor specific apoptosis as a globular multimer

Leviveld et al 2002 JBC

Purpose

To study the molecular mechanisms involved in Apoptin induced apoptosis

Identification of Apoptin-Associated Proteins by Identification of Apoptin-Associated Proteins by

Yeast Two-Hybrid SystemYeast Two-Hybrid System

APAP1

APAP2

Growth onSD/-Trp/-Leu/-His

-gal filter assay

APAP3

APAP4

Growth onSD/-Trp/-Leu/-His

-gal filter assay

APAP3 Identical to a Interferon induced IFP35

APAP2 An unknown gene with 6000b.p. with ten

ankyrin repeats

APAP4 Identical to Hippi/Hip-1 protein interactor

APAP1 Identical to DEDAF (Death Effector Domain-

Associated Factor) and human RYBP (Ring1

and YY1 Binding Protein)

APAP3 Identical to a Interferon induced IFP35

APAP2 An unknown gene with 6000b.p. with ten

ankyrin repeats

APAP4 Identical to Hippi/Hip-1 protein interactor

APAP1 Identical to DEDAF (Death Effector Domain-

Associated Factor) and human RYBP (Ring1

and YY1 Binding Protein)

Cloning and characterization of Apoptin associated

protein 1 ( APAP1)

PART1

APAP1 is identical to DEDAF (Death Effector Domain-Associated Factor) and RYBP (Ring1 and YY1 Binding Protein)

APAP1

226 a.a.

Peptide structure of APAP1

Zinc finger NLS NLS

KKEKK KKTKPK

CSVCTFRNSAEAFKCSIC

Purpose

Demonstration of in vivo and in vitro binding of Apoptin and APAP1

GST pull down of Apoptin and APAP1

pGEX/

Apoptin

pET32a(+)/

APAP1

E.coli transformation( BL-21)

IPTG induction

GST pull down

SDS PAGE

Western Blot( anti-His detection)

4oC sonication

Apoptin associate with APAP1 in vitro

Co-immunoprecipitation of Apoptin and APAP1

cell harvest, IP/ -GFP

DNA transfection

24hrs, 37oC

SDS-PAGE

Blot -Flag and -GFP

HeLa cells

GFP

pEGFPC2/

Apoptin

pRK5F/

APAP1 Flag

Apoptin associate specifically with APAP1 in vivo

Interaction domain mapping of Apoptin and APAP1

Small scale LiAC/ yeast transformation

Growth on SD-Trp-Leu-His medium

colony lift filter assay

pAS2-1/

Apoptin

BD

pACT/

APAP1 mutant

AD

Cellular localization of Apoptin and APAP1

16 hrs after transfection

psRed c1/

APAP1

RFPGFP

pEGFPC2/

Apoptin

HEL cells HeLa cells

DNA transfection

cells fix and DAPI stain

Fluororescent microscope

Apootin and APAP1 are colocalized at the nucleus region of HeLa cells

Purpose:

Involvement of APAP1 in Apoptin induced Apoptosis

Apoptotic Assay of Apoptin and APAP1

DNA co-transfection

48hrs, 37oC

GFPpEGFPC/

pRK5F/APAP1

Flag Flag

pRK5F/Apoptin

or

HEL cells HeLa cells

cells fix, PI stain and fluorescent microscope

APAP1 expression level in human tumor cell lines

Human tumor & normal cell lines

total cell protein extraction

SDS PAGE

Blot - APAP1 antibody

Normal cells Tumor cells

Higher APAP1 expression level in human tumor cell lines

summary

1. Apoptin associated specifically with APAP1 in

vitro and in vivo

2. Binding of Apoptin to APAP1 is localized to the region of a.a. 1-59 of Apoptin

3. APAP1 and Apoptin are colocalized at the nucleus region of HeLa cells

4. APAP1 induces apoptosis in both HeLa and HEL cells

5. Apoptin induced apoptosis is probably mediated by APAP1 through DED domain

6. Higher APAP1 expression level were observed in human tumor cell lines than normal cells.

Lower APAP1 expression level in human tumor cell lines

Apoptosis

APAP1

APAP4 APAP4

APAP1

AP

AP

1

AP

AP

1

AP

AP

4

Tumor cells

P

ApoptinP

PAPAP4

APAP1 AP

AP

1

AP

AP

1

AP

AP

1

APAP4

APAP4

APAP1

APA

P4

APAP4

AP

AP

4

APAP4AP

AP

4 AP

AP

1

APAP1

APA

P4APAP4

AP

AP

4

APAP4AP

AP

4

Apo

ptin

AP

AP

1

Cell survival

Normal cells

Apoptin

Ap

op

tin

Study and Characterization of APAP1/DEDAF in zebrafish development

Peptide structure of Z-APAP1

zAPAP1 257 a.a.

Zinc finger

GFWDCS VCTFRNSAEA FKCSIC

Actin

ZAPAP1

ZRYBP

PCR Amplification of APAP1 of zebrafish cDNA

Tissue Scan of Zebrafish cDNA

ZAPAP1

Actin

APAP1 siRNA increases the abnormal frequency of abnormal embryo