Biochemical techniques for detection of cell death - … documents... · Biochemical techniques for detection of cell death ... Measuring Apoptosis in Cell Culture: ... gel electrophoresis

Biochemical techniques for Biochemical techniques for detection of cell deathdetection of cell death

Boris ZhivotovskyBoris Zhivotovsky

Institute of Environmental Medicine, Institute of Environmental Medicine, KarolinskaKarolinska InstitutetInstitutet, Stockholm,, Stockholm,

SwedenSweden

Features of Apoptosis and NecrosisFeatures of Apoptosis and NecrosisFeatures of Apoptosis and Necrosis

CharacteristicsCharacteristics ApoptosisApoptosis NecrosisNecrosisStimuli Physiological or Pathological Pathological (injury)Occurrence Single cells Groups of cellsReversibility Limited LimitedCellular levelCellular levelCell shape Shrinkage and formation Swelling and later

of apoptotic bodies disintegrationAdhesion between cells Lost (early) Lost (late)Phagocytosis by other cells Present AbsentExudative inflammation Absent Present

Cellular organellesCellular organellesMembranes Blebbing Blebbing prior to lysisCytoplasm Late-stage swelling Very early swellingMitochondrial permeabilitytransition Present PresentNucleus Convolution of nuclear Disappearance

outline and breakdown (karyolysis)(karyorrhexis)

Features of Apoptosis and Necrosis (cont.)

Features of Apoptosis and Necrosis Features of Apoptosis and Necrosis (cont.)(cont.)

Biochemical levelBiochemical levelGene activation Present (?) Absent (?)Requirement for protein synthesis Present (?) Absent (?)Lysosomal enzyme release Present PresentActivation of non-lysosomal enzymes Present PresentActivation of caspases Present AbsentCleavage of specific proteins Present ?Changes in cytoskeleton Present PresentLevel of ATP required High Low Bcl-2 protection Present PresentNuclear chromatin Compactization in uniformly Clumping not

dense masses sharply definedDNA breakdown HMW and internucleosomal RandomizedRNA degradation Present ?Phosphatidylserine exposure Present Present

(?)- This feature is not a universal event or there are conflicting reports.

Measuring Apoptosis in Cell Culture:the inherent problem of asynchronyMeasuring Apoptosis in Cell Culture:Measuring Apoptosis in Cell Culture:the inherent problem of asynchronythe inherent problem of asynchrony

Start

CellTransduction Execution

Time

1

2

3

4

5

6

Percoll fractionation of apoptotic lymphocytes

PercollPercoll fractionation of apoptotic fractionation of apoptotic lymphocyteslymphocytes

Necrotic

Viability(% of total)

Necrotic

30%

40%50%

60%

70%80%94%

Viable

Viable

Pre-apoptotic

Apoptotic

3.4

4.9

92.0

91.689.4

65.2

6.4

14.9

1.9

1.622.6

78.5

% Nuclearfragmentation

ØØ Are you studying apoptosis or necrosis?Are you studying apoptosis or necrosis?- test for general cytotoxicity (LDH, MTT, etc)

Ø Are you studying cell population, individualAre you studying cell population, individualcells or clinical material (sections, biopsies,cells or clinical material (sections, biopsies,etc)?etc)?

Analysis of plasma membrane changesAnalysis of plasma membrane changesAnalysis of plasma membrane changes

1. Lactate dehydrogenase (LDH) activity (transformation oftetrazolium, yellow, to formazan, red)

§ LDH, a stable cytosolic enzyme, is released upon cell lysis and can therefore beused as a marker for cell death

§ Release can be measured spectrophotometrically (max abs. at about 500 nm)

§ The amount of enzyme activity correlates to the number of damaged cells(both apoptotic and necrotic)

Analysis of plasma membrane changesAnalysis of plasma membrane changesAnalysis of plasma membrane changes2. Live-dead cell assay (staining with calcein AM and

ethidium homodimer-1)

Ø Calcein acetoxymethyl ester is a membrane-permeant esterase substrate, whicheasily stains living cells. Dead cells cannot convert Calcein-AM to its fluorescentsubstrate

Ø Ethidium homodimer-1 (EH-1) enters dead cells through deteriorating cellularmembranes and binds DNA and RNA

Human neuroblastoma cells treated with camptothecin.Cells were viewed with fluorescein (emissionat 530nm) and rhodamine (emission at 590nm)optics.

Calcein-positive cells (green fluorescence)indicate healthy cells with an intact membrane,whereas ethidium homodimer-1-positive cells(orange fluorescence) represent dead orseverely damaged cells

Analysis of plasma membrane changesAnalysis of plasma membrane changesAnalysis of plasma membrane changes

A

B

Annexin V-Alexa 568 (red)BOBO-1 (green)

Annexin V-Fluo (green)PI (red)

3. Staining with Annexin V, followed by (A) fluorescence orconfocal microscopy, or (B) FACS (co-staining withpropidium iodide)

Analysis of mitochondrial integrityAnalysis of mitochondrial integrityAnalysis of mitochondrial integrity

1. Staining with MitoTrackerRed and anti-cytochrome c Abs

U1285

U1810

Control γ-irradiation

NBM RARS RARS+G-CSF

Analysis of mitochondrial integrityAnalysis of mitochondrial integrityAnalysis of mitochondrial integrity2. Staining with Rhodamine 123 or JC-1 or TMRE, followed

by confocal microscopy analysis

Staining of rat cortical astrocytes by rhodamine 123

Potential-dependent accumulation of the cationic dye in mitochondria results in a relatively weakfluorescence signal due to self-quenching (left panel).Dissipation of the mitochondrial membrane potential by the uncoupler FCCP is marked by increasing fluorescence (middle panel) and subsequent redistribution of the dye throughout the cell (right panel)

NIH 3T3 fibroblasts stained with JC-1, showing the progressiveloss of red J-aggregate fluorescence and cytoplasmic diffusion ofgreen monomer fluorescence following exposure to hydrogen peroxide

Analysis of mitochondrial integrityAnalysis of mitochondrial integrityAnalysis of mitochondrial integrity3. Staining with Rhodamine 123 or JC-1 or TMRE,

followed by FACS analysis

Cisplatin, 40 µM

IR, 8Gy

20 h 24 h

24 h 36 h

TMRE-FL

Analysis of mitochondrial integrityAnalysis of mitochondrial integrityAnalysis of mitochondrial integrity4. Measurement of the disruption of mitochondrial

membrane potential and superoxide generation

U1285

U1810

Control STS8 Gy γ-radiation

DiOC6(3) (∆Ψm) HE

E

th (

supe

roxi

de g

ener

atio

n)

zVAD-fmk+STS

Analysis of mitochondrial integrityAnalysis of mitochondrial integrityAnalysis of mitochondrial integrity5. MTT assay to measure changes in mitochondrial membrane potential

(conversion of the yellow, water-soluble, tetrazolium MTT to the blue, water insoluble formazan) (measured with an optical density reader)

Ø Conversion is catalyzed by cellular mitochondrial dehydrogenases,which is proportional to the number of surviving cells

6. Staining with anti-Apo 2.7 Abs followed by FACS

Analysis of metabolic changesAnalysis of metabolic changesAnalysis of metabolic changes

1. Intracellular acidification (SNARF-1, acetoxymethyl ester, shifts the color from red to yellow)

2. Changes in intracellular Ca2+ concentration (fura-2, fluo-3 or fluo-4)

3. Changes in cellular oxidative activity:a) Dihydroethidine (mitochondria)b) 5-(and-6-)-carboxy-2’ 7’-dichlorodihydro-fluorescein diacetate(carboxy-H2DCFDA) or RedoxSensorRed (cytosol)

c) cis-parinaric acid (lipids)d) Monochlorobimane, or FluoReporter (glutation depletion)

Analysis of protease activationAnalysis of protease activationAnalysis of protease activation

1. Cleavage of fluorogenic or chromogenic substrates, which are specific for different proteases

A

Flu

ores

cenc

eun

i ts(F

U )

5000

10000

15000

20000

0 10 20 30

Control 2hFas 2h

Time (min)

Am

oun

tA

MC

(pm

ole

s)

0

200

400

600

800

0 10 20 30

Control 2hFas 2h

Time (min)

B C

DE

VD

ase

activ

itypm

ole

s/m

in

0

5

10

15

20

Control 2h Fas 2h

Fluoroscan or spectrophotometer


2. FACS or fluorescent microscopy analysis of cleavageof fluorogenic substrates (PhiPhiLux)

Control aFas Ab


3. FACS (A) or fluorescent (B) or confocal microscopyanalysis of active caspases using mAbs

DAPI Active Caspase-3

A B


A

B

p32p20p17

p116p85

0 15 30 45 60 75 90 min

Caspase-3 processing (A) and PARP cleavage (B) analysis by immunoblotting

Cytometric Bead ArrayHuman apoptosis kit for detectionof cleaved PARP, active caspase-3 and Bcl-2

4. Western-blot or FACS analysis of targeted proteins

Analysis of protease activationAnalysis of protease activationAnalysis of protease activation5. Affinity labeling of active caspases with biotin-labeled

tetrapeptidesCo

ntrol

etopo

side

a-Fa

ssta

uros

porin

e

Activecaspases

Casp-3 Casp-6

Caspases were labeled with biotin-YVAD-amk andvisualized by 1D or 2D affinity blots

High

Low

MW

Acidic BasicpH

Analysis of protease activationAnalysis of protease activationAnalysis of protease activation6. Caspase activity measured by staining with Abs

against cleavage product of keratin

Spontaneous apoptosis in tissues from mouse and rat.Staining with M30-biotin Abs, counterstaining with Hematoxilin

Analysis of protease activationAnalysis of protease activationAnalysis of protease activation7. Cleavage of gelatin or other substrates in the gel

followed by gel staining

MWkDa

Cnt

Treatment

A0A10 B C D E

Analysis of endonucleasesAnalysis of Analysis of endonucleasesendonucleases

1. Direct DNA-degrading activity in SDS-PAGE

2. Incubation of isolated nuclei with nuclear protein extracts followed byelectrophoresis

3. Cleavage of plasmid DNA with nuclear protein extracts

TreatmentCnt A0 A10 B C D E

TreatmentCnt A0 A10 B C D E

1 2, 3

Analysis of DNA fragmentationAnalysis of DNA fragmentationAnalysis of DNA fragmentation

1. Diphenylamine reaction (quantitative assay)

(Burton, 1956)

Recipe:100 ml glacial acetic acid1.5 g diphenylamine 1.5 ml concentrated sulfuric acid0.5 ml 16 mg/ml acetaldehyde stockPrepare just before use

Read absorbance at 600nm.Express results as thepercentage of DNA fragmented

% fragmented DNA = x100Absorb. supernatant

Absorb. Supernatant + pellet


2. Comet assay

Illustration showing comets obtained from an untreated cell (A)and a cell with damaged DNA (B) with neutral comet assay


Modified from Leninger et al., 1993Principles of Biochemistry

Twochromatides(2x10 coils

One coil(30 rosettes)

One rosette(6 loops)

One loop(50 x 103 bp)

30 nm Fiber

“Beads-on-a-string” form of chromatin

DNA

Nuclearscaffold

4. Pulse-field gelelectrophoresis

3. Conventionalgel electrophoresis

Analysis of DNA fragmentationAnalysis of DNA fragmentationAnalysis of DNA fragmentation5. 3´-OH-end labeling (Klenow polymerase, followed by

gel electrophoresis and autoradiography)6. ISNT (In situ nick translation mediated by DNA

polymerase I)

Analysis of DNA fragmentationAnalysis of DNA fragmentationAnalysis of DNA fragmentation7. TUNEL (terminal deoxynucleotidyl transferase mediated dUTP

nick end labeling)(biotin-dUTP, digoxigenin-dUTP, or FITC-dUTP)a) Single cells - analysis with microscopy (A) or by FACS (B)

Autofluorescence

TUNEL without enzyme

TUNEL reaction

Control TreatmentA B


nick end labeling)(biotin-dUTP, digoxigenin-dUTP, or FITC-dUTP)a) Single cells - analysis with microscopy

Jurkat

U1285

U82

U1810

PITUNEL

Hoechst


nick end labeling)(biotin-dUTP, digoxigenin-dUTP, or FITC-dUTP)b) Tissue sections - analysis with microscopy

Identification of apoptosis-related genes by techniques that analyze gene

expression

Identification of apoptosisIdentification of apoptosis--related genes related genes by techniques that analyze gene by techniques that analyze gene

expressionexpression

1. Comparative approaches, including gene microarray analysis, serial analysis of gene expression, and differential display provide global information about expression levels.

2. Subtractive approaches like complementary DNA representational difference analysis (cDNA RDA) and suppression subtractive polymerase chain reaction identify a focused set of differentially expressed genes.

3. A retroviral insertion mutagenesis approach identifies apoptosis regulatory genes.

Methods for the Detection of ApoptosisMethods for the Detection of ApoptosisMethods for the Detection of Apoptosis

RNase protection assay for the detection of the expression of mRNA species for caspases, Bcl-2-related proteins, Death receptors, Death ligands, Signal proteins and Inhibitor proteins (IAPs)

Pro-caspase-2

Pro-caspase-3

Pro-caspase-7

Pro-caspase-8

Pro-caspase-9

RNase protection assay

10a

8436

52

71

9L32

G3PDH

pro-caspase

NSCLC

U17

52U

1810

H23

H12

5H

661

SCLC

U12

85U

1690

U20

50H

8 2H

5 92

He L

aH

L 60

Jurk

at

Prob

e s

y eas

ttR

NA

U17

52

U18

10

H23

H12

5

H15

7

H66

1

U28

5

U16

90

U20

50

H82

H59

2

NSCLC SCLC

Verification

Methods for the Detection of ApoptosisMethods for the Detection of ApoptosisMethods for the Detection of ApoptosisRNase protection assay for the detection of the expression of mRNA species for caspases, Bcl-2-related proteins, Death receptors, Death ligands, Signal proteins and Inhibitor proteins (IAPs)

RNase protection assay3

2

1

0

fold

OD

3

2

1

0

fold

OD

3

2

10

fold

OD

4

56

7

SCLS NSCLS

cIAP-1

cIAP-2

XIAP

Verification

Assays to examine the requirement of putative cell death genes

Assays to examine the requirement of Assays to examine the requirement of putative cell death genesputative cell death genes

1. Targeting gene deletions by means of homologous recombination (time consuming, technically demanding, and expensive)

2. Transfection of antisense oligonucleotides (ineffective if the targeted gene is expressed at high levels)

3. siRNA approach

4. A retroviral insertion mutagenesis approach identifies apoptosis regulatory genes

5. Overexpression of genes

Identification of protein interactions by yeasttwo-hybrid screening and coimmunoprecipitation

Identification of protein interactions by yeastIdentification of protein interactions by yeasttwotwo--hybrid screening and hybrid screening and coimmunoprecipitationcoimmunoprecipitation

X

Gal4(1-147)

UASG Gal1-lacZ

DNA-binding domain hybrid

UASG Gal1-lacZ

Activation domain hybridsencoded by a library

Y1-nGal4(768-881) X

Gal4(1-147)

UASG Gal1-lacZ

Interaction between DNA-binding domainhybrid and a hybrid from the library

YiGal4(768-881)

Method is based on the Gal4 or LexA systems

Expressionof fusionproteins

Chooseyeaststrain

“Pilot” screentransformation

Assayauto-

activation

Largescale

librarytransformation

PickHis3

Assay forLacX

PrepareDNA

Sequenceinsert

Assay specificityoutside two-hybrid

Assay specificityinside two-hybrid

Assay biologicalrelevance of interaction Co-IP

Constructionof binding

domain-targetfusion protein

Librarychoice

“Genetic”positives

1.1. Identification of protein interactions by yeast twoIdentification of protein interactions by yeast two--hybrid screeninghybrid screening(Field, S. and Song, O. Nature, 1989: 340, 245-246).

2.2. Identification of protein interactions by bacterial twoIdentification of protein interactions by bacterial two--hybrid screeninghybrid screening

(Joung, JK, Ramm, EI, and Pabo, CO. Proc. Natl. Acad. Sci, USA 2000; 97, 7382-7387).

3.3. A combined yeast/bacteria twoA combined yeast/bacteria two--hybrid systemhybrid system(Serebriiskii IG, et al., Mol. Cell Proteomics, 2005: 4.6, 819-826).

4.4. Mammalian twoMammalian two--hybrid assay for detecting proteinhybrid assay for detecting protein--protein interactionsprotein interactions

(Lee, JW, and Lee, S.-K. Meth. Mol. Biol. 2004, 261: 327-336).

Identification of proteins involving in cell deathby fractionation

Identification of proteins involving in cell deathIdentification of proteins involving in cell deathby fractionationby fractionation

HeLa Cell S-100

SP-Sepharose column

Flow -through Bound

Hydroxyapatitecolumn

BoundElutionKPO4

Heparin-Sepharose

column

Elution

Superdex 20016/60 column

Elution

Mono Q 5/5column

Apaf-1

Elution

Apaf-3(Caspase-9)

Flow -through

Q-Sepharose column

BoundElution

Phenyl-Superose 10/10

column

Ammonium sulfate

Supernatant

Heparin-agarosecolumn

Elution

Elution


Elution

Mono Q 5/5column

Elution

Glycerol gradientcentrifigation

Elution

Ammonium sulfate

Phosphocellulose

Supernatant

Supernatant

Phenyl-Sepharose column

Elution


Elution

Mono Q and S5/5 column

Apaf-2(Cytochrome c)

Elution

Identification of proteins involving in cell deathby fractionation

Identification of proteins involving in cell deathIdentification of proteins involving in cell deathby fractionationby fractionation

1. Protein fractionation, following western blot analysis and/orMass spectroscopy, Matrix-assisted laser desorptionionization post-source decay (MALDI-PSD), etc.

2. Proteomic approach

Cell-free systems to investigate cell deathCellCell--free systems to investigate cell deathfree systems to investigate cell death1. Cytoplasmic extracts from mitotic chicken hepatoma cells were found

to induce chromatin condensation and DNA cleavage reminiscent ofapoptosis in exogenously added nuclei

(Lazebnik, Y. et al., J. Cell Biol., 1993; 123, 7-22).

2. These extracts were used to identify a protease activity capable to cleave protein substrates during apoptosis(Lazebnik, Y. et al., Nature, 1994; 371, 346-347).

3. Cytoplasmic extracts of “aged” Xenopus eggs were found to induce apoptotic changes in nuclei added to these extracts(Newmeyer, DD. et al., Cell, 1994; 79, 353-364).

4. Cytoplasmic S-100 extracts were used to reproduce aspects of the apoptotic program in vitro. This program is initiated by addition of dATP(Liu et al., Cell, 1996; 86, 147-157).

Methods for the Detection of Apoptosis

Methods for the Detection of Methods for the Detection of ApoptosisApoptosis

In vivoIn vivo measurementsmeasurements

Methods for the Detection of Apoptosis

Methods for the Detection of Methods for the Detection of ApoptosisApoptosis

Staining with Annexin V labeled with 99mTc, followed by radionuclide imaging

In vivo imaging using a near infrared fluorescent-labelled(NIRF) annexin V (complex of annexin V with Cy5.5)

In vivoIn vivo imaging using a near infrared fluorescentimaging using a near infrared fluorescent--labelledlabelled(NIRF) (NIRF) annexinannexin V (complex of V (complex of annexinannexin V with Cy5.5) V with Cy5.5)

Visible light image ofimplanted tumors

Expression of DsRed2 in tumor (red fluorescence channel)

Near-infrared signal measured in tumors after injection ofCy-annexin (75 min)

Near-infrared signal measured in tumors after injection ofCy-annexin (20 h)

This method requires fluorescent endoscope an a minimally invasive fashion

Non-invasive detection of apoptosis using magnetic resonance imaging and a targeted contrast agent

NonNon--invasive detection of apoptosis using magnetic invasive detection of apoptosis using magnetic resonance imaging and a targeted contrast agentresonance imaging and a targeted contrast agent

C2 domain of synaptotagmin I is conjugated with superparamagnetic iron oxide (SPIO) nanoparticles. This complex binds to anionic phospholipids in the plasma membranes

MR images of a tumor in adrug-treated mouse followinginjection of C2-SPIO

Images obtained by subtractingthe post-contrast images fromthe image acquired beforeinjection of C2-SPIO

0 11 47 77 107min

Non-invasive detection of apoptosis using magnetic resonance imaging and a targeted contrast agent

NonNon--invasive detection of apoptosis using magnetic invasive detection of apoptosis using magnetic resonance imaging and a targeted contrast agentresonance imaging and a targeted contrast agent

A recombinant luciferase reporter molecule, E.R.–DEVD–Luc–DEVD–E.R., is cleaved and restore the luciferase activity, that can be detected in living animals with bioluminescence imaging

E.R. E.R.

DEVD DEVD

Luciferase

E.R.

DEVD

LuciferaseE.R.

DEVD

DEVD

Luciferase

Cautions when assessing and characterizing cell death

Cautions when assessing and Cautions when assessing and characterizing cell deathcharacterizing cell death

Ø Irrespective of the insult, the time-course of cell deathmight be very fast

Ø Clearance of apoptotic cells (phagocytosis) is alsomight be fast, especially in vivo

Ø In any static analysis only a small fraction of apoptoticcells might be detected

Ø Some techniques are not selective for apoptosis andshould be use in combination, and other may not be assensitive

Cautions when assessing and characterizing cell death

Cautions when assessing and Cautions when assessing and characterizing cell deathcharacterizing cell death

Ø Damaged vs. dead cells. Which damage is irreversible?

Ø Dose matters! High dose = necrosis, Mild injury = apoptosis

Ø Apoptosis and necrosis may simply represent two extremesof biochemically overlapping cell death pathways

Ø Some forms of cell death contain features of both, e.g.apoptosis is sometimes accompanied by secondary necrosis

Ø Paraptosis, Anoikis, Autophagy- all have some features ofapoptosis

Ø If you prevent apoptosis you may not prevent cell death, butsimply shift the mode of death to necrosis

ReferencesReferencesReferences

1. Methods in Cell Biology. Vol. 46, Cell Death. (L.M.Schwartz and B.A. Osborn, eds)Academic Press, 1995.

2. Techniques in Apoptosis. A user’s guide. (T.G.Cotter and S.J.Martin, eds)Portland Press, 1996.

3. Journal of Immunological Methods, Vol.265. Special Issue: Evaluation of Apoptosis.(M.-L- Gougeon and H.Lecoeur, eds ). 2002.

4. Methods in Molecular Biology. Vol. 282. Apoptosis Methods and Protocols.(H.J.M.Brady, ed). Humana Press, 2004.

5. Zhivotovsky, B., Samali, A., and Orrenius, S. Determination of apoptosis andnecrosis. In Current Protocols in Toxicology. (M.D. Maines, ed).

1999: pp.2.2.1-2.2.34. J. Wiley & Sons, Inc., New York.6. Kaufmann, S.H., Kottke, T.J., Martins, L.M., Henzing, A.J., and Earnshaw, W.C.

Analysis of caspase activation during apoptosis. In Current Protocols in CellBiology. (J.S. Bonifacino, M. Dasso, J.B. Harford, J. Lippincott-Schwartz,K.M. Yamada, eds). 2001: pp.18.2.1-18,2,29. J. Wiley & Sons, Inc., New York.

7. Zhivotovsky, B., and Orrenius, S. Assessment of apoptosis and necrosis byDNA fragmentation and morphological criteria. In Current Protocols in CellBiology. (J.S. Bonifacino, M. Dasso, J.B. Harford, J. Lippincott-Schwartz,K.M. Yamada, eds). 2001: pp.18.3.1-18.3.23. J. Wiley & Sons, Inc., New York.

8. Gogvadze, V., Orrenius, S., and Zhivotovsky, B. Analysis of mitochondrial dysfunctionduring cell death. In Current Protocols in Toxicology. (M.D. Maines, ed).2004: pp.2.10.1-2.10.27. J. Wiley & Sons, Inc., New York.

Work in our group is supported by the grantsfrom:

Work in our group is supported by the grantsWork in our group is supported by the grantsfrom:from:

European Commission

Documents

Biochemical techniques for detection of cell death - … documents... · Biochemical techniques for detection of cell death ... Measuring Apoptosis in Cell Culture: ... gel electrophoresis