Cytometry of Apoptosis

Zbigniew Darzynkiewicz, M.D., Ph.D.

Brander Cancer Research InstituteNew York Medical College

Hawthorne, NY 10536

darzynk@nymc.edu

Detection of Mitochondrial Changes and Activation of Caspases and Serine proteases

HeNe laser

LSC Optical System

Scan Lens

CCD Camera

Objective Lens

Computer Controlled Stage

Scatter Sensor

Scanning Mirror

Dichroic Mirrors and

Optical Filters

Photomultipliers

Argon ion Laser

Violet Diode Laser

THRESHOLD CONTOUR

INTEGRATION CONTOUR

PERIPHERAL CONTOUR

BACKGROUND CONTOUR

nucleus

cytoplasm

Setting contours by LSC

Parameters measured by LSC (I)

• Fluorescence integrated per integration contour area • Maximal intensity of individual pixel (“maximal pixel”)• Integration area (number of pixels)• Perimeter of integration contour• Fluorescence integrated over a torus defined by peripheral contour (e.g. nucleus vs cytoplasm)• Backround fluorescence (automatically subtracted) • xy coordinates of maximal pixel (location on slide)• Computer clock time at the moment of measurement

Apoptosis - Falling off petals of the flower. Connotation of natural death

Apoptosis: Active cell death; “Cell suicide”

Necrosis: Accidental cell death; “Cell murder”

Role of Cytometry in Analysis of Apoptosis

• To identify and quantify apoptotic cells

• To distinguish apoptotic from necrotic cell death

• To study molecular mechanisms of apoptosis

Morphology of apoptotic and necrotic cells

Apoptotic

Reduced cell sizeChromatin condensationNuclear fragmentationCell organelles unchangedMembrane “blebbing”Formation of apoptotic bodiesApoptotic bodies shed offCell remnants phagocytizedCells detach from substrate

Necrotic

Cell and nuclear swellingVacuolization of cytoplasmPatchy chromatin condensationMitochondrial swellingPlasma membrane ruptureDissolution of chromatinAttraction of inflammatory cells

Confocal 3d images of nuclei from nonapoptotic (A) and apoptotic (B) cells stained with PI

A B

HL-60 cells, Necrosis

Bcl-2Bcl-XL

Bcl-XL Bcl-2

Ca+2

Ca+2

Bad

Raf-1

P

Bad Bad

Bad

PT Pore

BaxConductance

Pore

Bag-1

Bc

l-2

Bc

l-2

AIF

Bax

Bax

Bax Bax

Bax

Bax

Bax Bcl-2

CYTOCHROME C RELEASE

CASPASE-9 APAF-1 (CED-4)

OLIGOMERIZATIONof APAF-1

ATP,dATP

ACTIVATION OF CASPASE-9“Initiator caspase”

CARD

ACTIVATEDCASPASE-9

CASPASES-3,-6,-7

ACTIVATION OF CASPASES

INTRACELLULARDEATH SIGNALS

nucleusPARPPARP

p53

Smac/DIABLO

IAP

Smac/DIABLO

IAP

Main pathways of apoptosis

Signals Death ligands (TNF, TRAIL) Internal cell stress,DNA damage

Death Receptors Cytochrome C Release

Caspase activator FADD Apaf-1

Bid

Initiator caspase Caspase-8, Caspase-10 Caspase-9

Effector caspase Caspase-3, Caspase-6 or Caspase-7

ExecutionExecutionCleavage of Apoptosis Regulators

Cleavage of Housekeeping ProteinsDNA Fragmentation

Features of apoptosis measured byflow or laser scanning cytometry

Plasma membrane Change in permeabilityChange in asymmetry of phosphatidylserine

NucleusChromatin condensationNuclear fragmentationDNA fragmentation

OrganellesChange in mitochondrial membrane potential (m)

Other featuresCaspase activationPARP cleavageSer-proteases activationTransglutaminase 2 activation

LINKERDNA

HISTONE OCTAMER(NUCLEOSOME

CORE)

DNAELECTROPHORESIS

Distance betweencuts = multiplicity of

200 base pairs

200 bp

400 bp

600 bp

800 bp

ICAD

CAD

DNA cleavage

Apoptosis-induced DNA fragmentation

dUTP -biotynylated

TdT TdT

avidin-FITC

DNA cleavage

ICAD

CAD

Indirect labeling of DNA breaks

Apoptotic in situ DNA strand breaks

Colocalization: DNA and DNA strand breaks

Apoptosis – DNA strand breaks

Cell cycle phase specificity of apoptosis-induced DNA strand breaks is revealed by the bivariate DNA content

vs DNA strand breaks analysis

Apoptosis in vivo; variablity in frequency of DNA strand breaks

Early: Few break sites

Mid: Maximal number of break sites; no loss of DNA content

Late: Loss of DNA is apparent and, as a result, so is loss of breaks sites, presumably due to shedding of apoptotic bodies

Bcl-2Bcl-X L

Caspase 9

PARP

PARP

nucleus

Bcl-X L - Bcl 2

Bcl

-2

Ca+2

Ca+2

Bad

Raf-1

P

Bad Bad

Bad

PT Pore

Bax ConductancePore

p53

Caspase 3

Bag-1

Bcl

-2

Bcl

-2

APAF-1

AIF

Cytochrome C

Bax Bax

Bax

Bax

Bax

Bax Bax

Bax

Bax

Early event of apoptosis: translocation of Bax to mitochondria

Bax immunofluorescence, MCF-7 cells, CPT-treated

Translocation of Bax to mitochondria

N

Bax in cytoplasm and in nucleus

Bax in mitochondria

C

(diffuse) (punctate)

Translocation of Bax to mitochondria:Increase of maximal pixel of Bax immunofluorescence

0

10

20

30

40

50

60

Control CPT 24h CPT 48hDNA Content DNA Content DNA Content

CTRL CPT 24h CPT 48h

150 150 1500 0 0

G1 S G2M

Ba

x M

ax

Pix

el

G1 S G2M

Ba

x m

ax

pix

el

DNA content

Dissipation of the mitochondrial trans- membrane electrochemical

potential

Rhodamine 123 or carbo-cyanine dyes

“Aggregate” dyes (e.g. JC-1)

HL-60 CTRL HL-60 CPT 4h

Rh 123(rhodamine 123)

Green IntegralGreen Integral

Sca

tter

Inte

gra

l

Sca

tter

Inte

gra

l

HL-60 CTRL HL-60 CPT 4h

JC-1 (“aggregate” dye)(5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazolcarbocyanine iodide)

Green IntegralGreen Integral

Ora

ng

e In

teg

ral

Ora

ng

e In

teg

ral

Detection of caspases activation

• Cleavage of the death substrates [e.g.poly(ADP)-ribose polymerase (PARP)]

• Antibodies reactive with activated caspases

• Fluorogenic or chromogenic substrates

• Fluorochrome-labeled caspase inhibitors (FLICA)

Immunocytochemical detection of p-89 PARP

Immunocytochemical detection of PARP p89

PARP cleavage vs appearance of DNA strand breaksCPT-induced

PARP cleavage vs appearance of DNA strand breaks

TNF-induced

PARP cleavage vs DNA strand break apperance

PARP cleavage vs appearance of DNA strand breaks

Bcl-2Bcl-X L

Caspase 9

PARP

PARP

nucleus

Bcl-X L - Bcl 2

Bcl

-2

Ca+2

Ca+2

Bad

Raf-1

P

Bad Bad

Bad

PT Pore

Bax ConductancePore

p53

Caspase 3

Bag-1

Bcl

-2

Bcl

-2

APAF-1

AIF

Cytochrome C

Bax Bax

Bax

Bax

Bax

Bax Bax

Bax

Bax

Is collapse of the mitochondrial potential a prerequisite for cytochrome c release ?

A B

A B

~100% B+ will be A+

some B+ will be A-

Multiparameter analysis; cause - effect relationship

Cell attributes studied supravitally

Cell attributes that require cell fixation

Integrity of plasma membrane

Surface immuno-typing

Phosphatidylserine on cell surface

Transport through membrane

Intracellular pH

Oxidative stress (ROIs)

Mitochondrial potential

Level of glutathione

Activation of intracellular enzymes

Calcium flux and other ions

Cell cycle position, DNA ploidy

BrdU incorporation

DNA strand breaks

Chromatin condensation

Activation of NF-kappa B

Bax, cytochrome c translocations

Translocation of other (AIF, APAF-1) molecules

Caspase activation (e.g. PARP cleavage)

Immunodetection of intracellular proteins

Changes in cell morphology

Live cells Measure m

Fix cells Measure PARP

File # 1

File # 2

Merge file # 1 and # 2

Analyze the merged file

Strategy to detect correlation between the paramaters measured on live (mitochondrial potential) vs fixed (PARP cleavage) cells

by LSC

Mitochondrial potential vs PARP cleavage

Fluorochrome-labeled inhibitors of caspassesFLICA

FAM

FMK

VEID

Affinity labeling probes of caspase enzymatic center

INACTIVE CASPASE (ZYMOGEN)

ACTIVATED CASPASE (HETERO-TETRAMER)

BINDING OF FLICA

prodomain

A

B

C

DFLICA

Active center

Active center

FAM

FMKVEID

FAM-DEVD-FMK

VADCTRL CPT 3H CPT 4H

VEID

TNF+CHX 1h TNF+CHX 1.5h

VADCTRL

PI - fluorescence

FA

M-V

AD

-FM

K

100

0

104 PI - fluorescence

FA

M-V

AD

-FM

K

100

0

104

FAM-VAD-FMK FAM-VAD-FMK100 100

N

umbe

r of

cel

ls

N

umbe

r of

cel

ls0 0

Control

Control

3h CPT

3h CPT

D

C

A

B

Caspases activation during apoptosis induced by CPT

Green Max Pixel

Cel

l fre

qu

ency

0

100

100

1 2

Protection of FAM-VAD-FMK binding by cell pre-exposure to z-VAD-FMK

A B

Caspases activation detected by FAM-VAD-FMK binding

FAM-VAD-FMK binding; fixed cells

FA

M-V

AD

-FM

K

flu

ore

scen

ce

PI fluorescence

Concurrent cell staining with FAM-VAD-FMK and PI

Time (h)

control 2h 3h 4h 5h

A

B C

D

B

A

DCC

ell n

um

ber

(%

)P I f l u o r e s c e n c e

100

0

104 104104 104 104

0 2 3 4 5

100

FA

M-V

AD

-FM

K

flu

ores

cen

ce

0 0000

100 100100100

Kinetics of FAM-VAD-FMK and PI binding

Caspase activation - cell cycle specificity

Effect of TNF on HL-60 cells

PI, INT

FA

M-V

AD

-FM

K;

MP

X

0

a b

103

103100

1000

FA

M-V

AD

-FM

K;

INT

FAM-VAD-FMK; INT

G1

S G2/M

A

BC

D

Propidium Iodide

FA

M-V

AD

-FM

KDistinction between apoptotic and necrotic cells based on caspases activation (FAM-VAD-FMK binding) vs exclusion

of PI

Ctr Parthenolide -treated

HL-60 cells

Annexin V, Max Pix

FA

M-V

AD

-FM

K,

Max

Pix

Annexin V, Max Pix

Correlation between caspase activation and Annexin V binding

FA

M-V

AD

-FM

K

flu

ores

cen

ce

P I f l u o r e s c e n c e

P I f l u o r e s c e n c e

FA

M-V

AD

-FM

K

flu

ores

cen

ce

FA

M-V

AD

-FM

K

flu

ores

cen

ce

FA

M-V

AD

-FM

K

flu

ores

cen

ce

FA

M-V

AD

-FM

K

flu

ores

cen

ce

100100

100100

100

0

0 0

0 0

104104

104104

104

3 %

2 %

4 % 9 %21 % 20 %

25 %

2 %

2 %

2 %

9 % 7 % 51% 10 %

2 %

6h3h

Stathmo-apoptosis; arresting progress of apoptotic process

Not arrested

Arrested

FA

M-V

AD

-FM

K

104

1040Propidium iodide

Control

A

C

D

P r o p i d i u m i o d i d e

B

FA

M-V

AD

-FM

K

104

1040

104

1040

104

1040

FA

M-V

AD

-FM

K

104

1040

TNF FLICATNF + FLICA

CPT + FLICA CPT FLICA

Stathmo-apoptosis ARRESTED NOT ARRESTED

A

B C

D

FA

M-V

AD

-FM

K

flu

ores

cen

ce

10 0

48h 72h

Control

Ap-1%

Ap-13% Ap-23%

Ap-39%Ap-24%

P I f l u o r e s c e n c e

0

0

FA

M-V

AD

-FM

K

flu

ores

cen

ce

FA

M-V

AD

-FM

K

flu

ores

cen

ce

0 0102

0

FA

M -

VA

D -

FM

K

flu

ores

cen

ceF

AM

-VA

D-F

MK

fl

uor

esce

nce

102

102 102

102

P I f l u o r e s c e n c e

10 0

10 0 10 0

10 0

Stathmo-apoptosis

Apoptotic MCF 7 cells prevented from detachment

Activation-induced apoptosis of lymphocytes

Caspases activation detected by FLICA

PI fluorescence

FA

M-V

AD

-FM

K

CTR PHA PHA+ONC

AA DA

B BB

DD

CCC

A

B C

D

0 24 48 72

0 0

Time (h)

4040

0 0

6060

0

60 Time (h)

CA

I (%

)C

AI

(%)

CA

I (%

)

2-CdA

Control

Subtracted CAI

Patient 1Patient 2

Patient 3 Patient 4

0 24 48 72

0 24 48 72

0 24 48 72

0 24 48 72

Patient 5

Detection of serine-proteases activation

• 5(6)-carboxyfluoresceinyl-L-phenylalanine chloromethyl ketone (FFCK), analog of N-tosyl-L-phenylalanine chloromethyl ketone (TPCK)

• 5(6)-carboxyfluoresceinyl-L-leucyl chloromethyl ketone (FLCK), analog of N-tosyl-L-leucyl chloromethyl ketone

FLISP (Fluorochrome-labeled inhibitor of Ser-proteases)

Asp-102

His-57

Ser-195

FAM

F (Phe)

CMK

Imidazole Alkylated imidazole

Active chymotrypsin Covalent binding of FLISP

Labeling of active enzyme center of Ser-proteases with FLISP

CPT-induced apoptosis of T-24 cells - Texas Red-FCK vs DAPI

Propidium iodide 104104104 104

FF

CK

FF

CK

F

LC

K

F

LC

K

FFCK FLCK FFCK FLCK

100

100 100 100 100

100 100 100

00 0 0

Control 3h CPT

Control

Control

Control3h CPT 3h CPT

3h CPT

N

umbe

r of

cel

ls

0 0 00

Activation of Ser-proteases reactive with FFCK and FLCK during apoptosis of HL-60 cells induced by CPT

SR-VAD-FMK100

0

F

FC

K

0

100

r = 0.72

Activation of caspases and Ser-protease(s) (FFCK-reactive) in HL- 60 cells 3 h after

addition of CPT

Activation of caspases and Ser-proteases

SR-VAD-FMK /FFCK

F

LC

K

SR-VAD-FMK100

0

100

r = 0.82

Activation of caspases and Ser-protease(s)

(FLCK-reactive) in HL- 60 cells

3 h after addition of CPT

FFCK vs. VAD (Casewise MD deletion)

VAD = 1.6472 + 1.0007 * FFCK

Correlation: r = .98609

FFCK

V

AD

0

15

30

45

60

75

0 10 20 30 40 50 60 70

FA

M-V

AD

-FM

K

FFCK

r = 0.98

FLCK vs. VAD (Casewise MD deletion)

VAD = 5.1191 + 1.0204 * FLCK

Correlation: r = .98178

FLCK

V

AD

5

15

25

35

45

55

65

0 10 20 30 40 50 60 70

FLCK

FA

M-V

AD

-FM

K

r = 0.982

Correlation between activation of caspases (FAM-VAD-FMK) and Ser-proteases during apoptosis of HL-60 cells induced by CPT

r = 0.986

SR-VAD-FMK (Int)

FF

CK

(In

t)

0

100

SR-VAD-FMK (Int)

FF

CK

(In

t)

0100

100

100

1000

Apoptosis of HL-60 cells induced by Onconase

FFCK vs SR-VAD-FMK binding

Ctrl Onc

100

SR-VAD-FMK (Int)

FL

CK

(In

t)

0 100

100

SR-VAD-FMK (Int)

FL

CK

(In

t)

0100

100

Apoptosis of HL-60 cells induced by Onconase

FLCK vs FAM-VAD-FMK binding

PI fluorescence

FA

M-V

AD

-FM

K

FF

CK

FL

CK

Ctrl Ctrl Ctrl

Onc Onc Onc

Activation of caspases and Ser-proteases during apoptosis of HL-60 cells induced by Onconase

205

112

70

52.4

Con

trol

FFC

KFF

CK

+TN

F

Con

trol

FLC

K

FLC

K+T

NF

Proteins covalently reactive with FFCK and FLCK(anti-fluorescein Ab)

Brander Cancer Research Institute at NYMC, Valhalla,

NY

E. Bedner

W. Gorczyca

J. Grabarek

G. Juan

X. Li

P. Pozarowski

P. Smolewski

F. Traganos

Z. Darzynkiewicz

Immunochemistry Technologies, Bloomington NM

B.W. Lee

G. Johnson

Activation of transglutaminase Tissue transglutaminase 2 (TGase 2)

G-protein signalling; protein crosslinking

• Cell resistance to detergents

• Attachment of fluorescinated cadaverine

A B

C D

*

*

* **

*

* *

TGase 2

Resistance to detergent (A,B)

Fluorescein-cadaverine binding (C,D)

SU

LF

OR

HO

DA

MIN

E

(pro

tein

)

100

100

0

A

D

100

1000

C100

1000

B

G1

G2M

S

DAPI; DNA content

TGase 2, cell resistance to detergent

FL

- c

adav

erin

e

DNA content100

0

100

FL

- c

adav

erin

e

DNA content100

0

100

G1G2MS

TGase 2; binding of fluoresceinated cadaverine

FAM-VAD-FCK vs PI FFCK vs PI FLCK vs PI