Gérard LIZARD - Inserm

EA7270 - Equipe ‘Biochimie du Peroxysome, inflammation et Métabolisme Lipidique’

Faculté des Sciences Gabriel

6, Bd Gabriel, 21000 Dijon - FRANCE

Caractérisation et méthodes d’études de la mort cellulaire

par cytométrie en flux

Université de Bourgogne

Gérard Lizard

Rabat, 29 novembre 2018

CytoMagh 2018

Two pathways of cell death leading to necrosis and apoptosis. At the top is shown a normal cell.

1A: Swelling. 1B: Vacuolization, blebbing, and increased permeability. 1C: Necrotic changes. ie,

coagulation, shrinkage, and karyolysis. 2A: Shrinkage and pyknosis. 2B: Budding and karyorhexis.

2C: Necrotic changes, ie, breakup into a cluster of apoptotic bodies.

Majno G & Joris I Am J Pathol 1995, 146: 3 - 15. Gérard Lizard,

ORGANELLES INVOLVED IN CELL DEATH

Organelles Apoptosis Autophagy Necrosis /

Necroptosis

- Mitochondria + +

(mitophagy) +

- Lysosomes + / - + + / -

- Endoplasmic-

reticulum (ER) + / - +

+ / -

- peroxisome ? +

(pexophagy)

?

Bröker LE et al. Clin Cancer Res 2005, 11: 3155-3162. Gérard Lizard,

4

Relations peroxysome / mitochondrie

Implication dans le contrôle de l’équilibre RedOx et l’activation de la mort cellulaire

Lismont C, Nordgren M, Van Veldhoven PP, Fransen M. Front Cell Dev Biol. 2015, 27;3:35.

Cell Death (initial concept)

Apoptosis apoptotic morphology

Active programmed cell death

Caspase-dependent cell death

• Mitochondrial pathway

(Associated or not with reticulum stress)

• Death receptor pathway

Necrosis necrotic morphology

Passive unprogrammed cell death

Classical /Canonical Necrosis

Cell Death Independent of Caspases

Kitanaka C & Kuchino Y Cell Death Differ 1999, 6: 508-515. Gérard Lizard,

Cell death

Apoptosis Autophagy Necrosis

Active programm cell death

Caspase-dependent

Type-1 cell death

Active programm cell death

Caspase-independent

Type-2 cell death

Active programm cell death

Caspase-independent cell death

Necroptosis (RIP1, RIP3)

Passive programm cell death

Caspase-independent cell death

Classical necrosis (primary necrosis)

Type-3 cell death

Secondary necrosis

Gérard Lizard,

AVAILABLE METHODS ALLOWING THE CHARACTERIZATION

OF APOPTOSIS, NECROSIS / NECROPOTOSIS, AND AUTOPHAGY

Gérard Lizard,

FUNCTIONNAL CRITERIA ASSOCIATED WITH APOPTOSIS,

NECROSIS, AND NECROPTOSIS

• Microscopy, flow cytometry, biochemistry

- enhanced permeability of cytoplasmic membrane (trypan blue, fluorescent probes, LDH)

- externalization of phosphatidylserine

double staining with AnnexinV /propidium iodide (PI) (or aminoactinomycine D (AAD))

to distinguish between normal (AnnexinV-/PI -), necrotic (AnnexinV+/PI+),

and apoptotic (AnnexinV+/PI-) cells

- Coloration SYTO16 - IP

- Sub-G1 peak (not present in normal and necrotic cells)

- loss of transmembrane mitochondrial potential: numerous fluorescent probes available.

Currently, DioC6(3) and JC1 are the most reliable.

- FLICA (fluorochromes labeled inhibitors of caspases): in situ identification of activated

caspases (does not permit to distinguish between necrosis and caspase-independent cell death)

- TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling): in situ identification

of internucleosomal DNA fragmentation

Gérard Lizard,

Electron microscopy A: Control. B: Cells treated by VP-16; apoptotic cells with condensed and perinuclear

chromatin are observed, cytoplasmic and nuclear membrane integrity are preserved as well as morphology of

mitochondria (arrow). C: Cells treated by NaN3; necrotic cells are characterized by a loss of integrity of

cytoplasmic and nuclear membranes, degradation of cytoplasm and chromatin.

Fluorescence microscopy after staining with Hoechst 33342 D: Control cells. Nuclei show regular contours. E:

Cells treated by VP-16; cell with fragmented (f) nucleus. F: Cells treated by VP-16; cell with condensed (c)

nucleus. G: Necrotic (n) cells observed under treatment by NaN3;

the nucleus is diffuse and irregular. (Lizard G et al. Cytometry 1995, 21: 275-283)

APOPTOSIS / NECROSIS: MORPHOLOGICAL CRITERIA

Gérard Lizard,

MORPHOLOGICAL CRITERIA ASSOCIATED WITH APOPTOSIS,

NECROSIS AND NECROPTOSIS

* Phase contrast microscopy: loss of refringence; loss or not of cell

adhesion?

* Brightfield microscopy: after staining with GIEMSA,…

* Fluorescence microscopy: Hoechst staining allows to easily distinguish

between normal, necrotic and apoptotic cells (nuclear criteria)

* Transmission electron microscopy (nuclear criteria)

* Flow cytometry: changes of light scatter properties on non-fixed cells

(FSC ↓ ; SSC more or less ↑ )

Control 7-keto

40 µg/ml

U937 G

Gérard Lizard,

http://www.amnis.com

Henery S et al. Apoptosis 2008, 13: 1054-1063

Annexin V-FITC / PI (Amnis technology)

Gérard Lizard,

Phosphatidylserine externalization : Annexin V-FITC / PI test

Apoptosis

Necrosis

(phosphatidylserines)

V A

N

Annexin V - FITC

PI (o

r 7A

AD

)

Control Treated

PI: propidium iodide

7 AAD: 7 amino actinomycin D

Viable (V)

Apoptotic (A)

Necrotic (N)

Annexine V PI Cells

Gérard Lizard,

Towards an Understanding of Apoptosis Detection by SYTO Dyes Donald Wlodkowic,1* Joanna Skommer,1 and Jukka Pelkonen1,2

1Institute of Clinical Sciences, Department of Clinical Microbiology, University of Kuopio, Kuopio, Finland

2Department of Clinical Microbiology, Kuopio University Hospital, Kuopio, Finland

Cytometry Part A 71A:61–72 (2007)

Necrotic cells

Viable cells Apoptotic cells

Gérard Lizard,

Nu

mb

er

of

ce

lls

Sub-G1

control

cholesterol 7ß-hydroxycholesterol

7-ketocholesterol

HUVECs

Lizard G et al. Am J Pathol 1996, 148: 1625-1638

Sub-G1 (FCM)

apoptosis

primary or secondary necrosis

Gérard Lizard,

0

20

40

60

80

100

0 6 12 18 24

Time of treatment (hours) % c

ell

s w

ith

dep

ola

rize

d m

ito

ch

on

dri

a

control

7-ketocholesterol (40 µg/ml)

7b-hydroxycholesterol (20 µg/ml)

Control 7-ketocholesterol 7-hydroxycholesterol

Mitochondrial potential (FCM)

Gérard Lizard,

Grabarek J & Darzynkiewicz Z Exp Hematol 2002, 30: 982-989

FLICA / FLISP (Microscopy, Flow Cytometry)

Gérard Lizard,

TUNEL (Flow Cytometry)

Gérard Lizard,

TUNEL (Amnis technology)

Gérard Lizard,

Lizard G et al., Arterioscler Thromb Vasc Biol 1999, 19: 1190-2000

Phase contrast microscopy, Hoechst 33342, GIEMSA, TUNEL

Apoptosis = TUNEL positive cells

Necrosis = TUNEL negative cells

Gérard Lizard,

BIOCHEMICAL CRITERIA ASSOCIATED WITH

APOPTOSIS, NECROSIS, AND NECROPTOSIS

* Analysis of the DNA fragmentation pattern on agarose gel

- Apoptosis: DNA ladder, multiple of 150-200 base pairs

(internucleosomal DNA fragmentation)

- Necrosis: no DNA ladder, smears

* Enzymatic activities (LDH)

* Electrophoresis on acrylamide gel and Western Blotting

Gérard Lizard,

Huang WC, Chen CL, Lin YS, Lin CF. Apoptotic sphingolipid ceramide in cancer

therapy.

J Lipids. 2011;2011:565316.

Anticorps anti-céramides

Hoechst

Cellule

normale

Cellule

pré-apoptotique

Cellule

apoptotique

Exp

res

sio

n d

e c

éra

mid

es

Stimuli apoptotique : génération de céramides

(détection par immunofluorescence)

MCF-7 MCF-7/c3

Prunet C et al. Cell Death Differ, 2001; J Biochem Mol Toxicol, 2005

Caspase-3/7

Caspase-8

Caspase-6

DYm without caspase-3

Oncosis

AIF, EndoG

cytochrome-C

Caspase-9

Caspase-3

Caspase-7 Caspase-8

Bid

t-Bid

Apoptosis

PARP

ICAD CAD

Necrosis/Oncosis = no caspase activation

Gérard Lizard,

Gérard Lizard,

CHARACTERIZATION OF AUTOPHAGY:

‘VISUAL’ CRITERIA

7KC (50 µM, 24 h)

Control

EtOH (0.1%)

0.5 µm

1 µm

1 µm

1 µm

1 µm

ap

ap

ap: autophagosome; apl: autophagolysosome

1 µm

apl

0.1 µm

A

B

C

D

E

F

G

H apl

0.1 µm

Gérard Lizard

CHARACTERIZATION OF AUTOPHAGY:

ULTRASTRUCTURAL CRITERIA – Transmission Electron Microscopy

CHARACTERIZATION OF AUTOPHAGY: BIOCHEMICAL CRITERIA Murine oligodendrocytes (158N) - autophagy and apoptosis (oxiapoptophagy)

Co

ntr

ol

EtO

H (

1%

)

α-c

yclo

dex

trin

(12

5 µ

M)

α-t

oco

ph

ero

l (4

00

µM

)

DH

A (

50

µM

)

7K

C (

50

µM

)

7K

C +

α-t

oco

7K

C +

DH

A

7K

C +α

-to

co+

DH

A

7β

-OH

C (

50

µM

)

7β

-OH

C +α

-to

co

7β

-OH

C +

DH

A

7β

-OH

C +

α-t

oco

+DH

A

24

S-O

HC

(5

0µ

M)

24

S-O

HC

+α

-to

co

24

S-O

HC

+D

HA

24

S-O

HC

+ α

-to

co +

DH

A

Caspase -3 (35 kDa)

Cleaved Caspase -3 (17 kDa)

PARP (116 kDa)

Cleaved PARP (89 kDa)

β-Actin (42 kDa)

LC3-I (18 kDa)

LC3-II (16 kDa)

Bcl-2 (26 kDa)

Ratio LC3-II/LC3-I 0.6 0.5 3.8 0.8 0.6 0.7 1.7 1.3 1.2 1.3 3.4 1.7 1.2 0.9

EtO

H (

0.1

%)

EtO

H (

1.1

%)

α-c

yclo

dex

trin

+ E

tOH

(0

.1 %

)

α-c

yclo

dex

trin

+ E

tOH

(1

.1 %

)

0.6 0.6 0.6 0.6 0.7 0.5 0.4

Gérard Lizard,

Apoptosis Necrosis Necroptosis

Phosphatidyl serine

externalization

(Annexin V positive + PI positive)

Mitochondrial depolarization

Random DNA degradation

RIP1 (and RIP3)

expression

Phosphatidyl serine

externalization

(Annexin V positive + PI negative)

Mitochondrial depolarization

Non Random DNA degradation

(DNA ladder)

Caspases activation

Gérard Lizard,

Autophagy

Oxiapoptophagy

Necroapoptophagy

LC3 conversion:

LC3I to LC3-II