The far side - the gamma-H2AX Focus Assay

Harry Scherthan

Bundeswehr Institute of Radiobiology affiliated to the Univ. of Ulm, D-80937 Munich, Germany

scherth@web.de

DSBs result from ionizing radiation and radiomimetic drugs

• Arise physiologically during DNA replication

• Are delivered as initiator of physiological recombination in

processes like V(D)J recombination & Meiosis.

DNA double-strand breaks (DSBs) are a

severe threat to genomic integrity

Physiological DSBs & gH2AX in mammalian meiosis

Scherthan

g-H2AX

NucleiMouse (image: H.S.)

RAP1

IR

ROS

MDC1

ATM (DNAPKcs)

NBN

RAD50

MRE11

BRCA1

RAD51

H2

DNA RepairChromatin modification

Apoptosis MDM2, p53, Bax

Cell cycle arrestCHK1, -2; CDC25A,B

Stress responseSAPK

HR

RPA

cABL

DNA PKKu70/80

NHEJP

P

M/R/NBN

53BP1

scherth@web.de

H2A.X

53BP1

DNA damage response - DDR

Histone H2A.X in chromatin context

DNA

DSB

Scully & Xie MR 2013

γH2AX forms a chromatin domain @ a DSB

DSB

NHEJ

based on Jeggo et al. 2011 Rad Onc

ss break=> replic. fork

stalling/collapse

IR-induced DSBs & repair pathway choice

IR-induced DSB

HDR

NHEJ protects premeiotic stem cells and meiosis from accumulation of DNA damage

Ku70-/- spermatogenesis

Ahmed, Sfeir, Scherthan J Cell Sci 2013

B

B

B

B

B

B

B

B

B

Ku70-/-WT S

S

XY

XYXY

XY

53BP1

γH2AX

Ku70-/- γH2AX cell death

XII

MI

DNA 53BP1

MEIOSIS: homol. recombination DSB repair during prophase I

Rad50,Mre11,Nbs1 (=MRN),

Com1

RPA, WRN, Tex14

Rad51, Dmc1, Hop2

52, 55/57

BLM/Sgs

Rad51, Rad54, Tid1Rap1

Rad54

Rad54

Spo11, MRN, Rec102,104,114

Zip1, Msh4,-5, Mlh1

ATM, Mec1/Tel1

g-H2AX, SCP3♂ mouse

g-H2AX Cytology

g-H2AX

Moens et al.

2002 JCS

Liebe et al. 2006 ECR

Spo11-/-

(no DSBs)

Spo11+/+

gH2AX

SCP3

Meiosis: IR-induced DSBs can modulate chromatin phosphorylation &

prophase I progression in cells without physiol. DSBs (Spo11-/-)

Spo11-/- IR

3Gy X-rays

Liebe et al. 2006 ECR

gH2AX

♀ SCP1, g-H2AX, DNA ♀ g-H2AX, RPA

Roig et al. 2004, Chromosoma

♂ g-H2AX, RPA, SCP3

XY body

♀ pachytene ♂ pachytene

g-H2AX localization @ pachytene

0

20

40

60

80

100

SC-assoc. g-

H2AX

g-H2AX / RPA g-H2AX / MLH1

class

%

Male

Female

Human meiosis: DNA repair and gH2AX

g-Photon 500 keV

a-Particle 4 MeV

DNA

IR induces chromatin & DNA damage

after Goodhead 1994, Aten 2003, Scherthan 2004

3Gy

ahighLET

Low LET

gH2AXDNAKinetochore

gH2AXDNARad51 (DSBs)

Method for quick detection of radiation-induced

damage: DNA-repair focus test

Sedelnikova et al. 2002 RR

Rothkamm & Löbrich 2003 PNAS

gH2AX focus enumeration for monitoring

ionizing radiation exposure

Rogakou et al. 1999, JCB

• IR-induced DSBs – ATM & DNAPKcs kinases phosphorylate the histone 2 variant H2A.X within minutes

around the site of a DSB to result in g-H2AX foci in the nucleus (Rogakou et al. 1998)

Repair half-time in the order of min –hours

In non-cycling tissue cells (e.g. skin) some DSBs

(complex DNA damage) may persist for days/weeks

! Absence of dephosphorylation can lead to focus

persistence wo DSB

Double-stranded DNA breaks (DSBs) & γH2AX

MRE11γH2AX

γH2AX53BP1

Irradiation: HR repair factors colocalize at DSBs

Lamkowski et al. 2014 PlosOne

100 Gy10 Gy1 Gy100 mGy10 mGy1 mGy100 µGy10 µGy

after Löbrich 2003

METHOD – in vitro blood irradiation

Method

1. Blood sampling

2. Leukocyte isolation

3. Fixation (store & transport [?])

4. Immunofluorescence staining

5. Analysis

IR

Scherthan et al. 2007 Wehrm .Mschr .

Focus Assay - Staining

Most labs: immediate staining after IR & incubation / blood cell isolation

1) Bring the cells to a me-silane coatedglass slide (cytocentrifuge; drying).

2) Fix with (1%-) 3.7% formaldehyde (!)3) Extract with TritonX1004) add primary antibody detection

Sample storage ? – yes (but) !

Ethanol fixed cells can be stored and shipped

- ! only compare with similarly treated (time & temp) controls.

- We use 70% Ethanol @ -20°C (Lassman et al. 2010 Nucl.Med.)

0

2

4

6

8

10

0 2 4 6 8

10

12

14

16

18

20

22

24

26

28

30

nu

cle

i %

n Foci/Zellkern

EthOH-Fixation PBCs 0Gy/1h fresh/old

LZ-RZ 28.07.frisch

LZ-RZ 25.08.frisch

LZ-HS 27.10.frisch

LZ-RZ 25.08.alt

Methodological pitfalls: Fixation/extraction determines the success of g-H2AX detection

0.15% TX100, 10 min. 0.5% TX100, 10 min.

kinetochoresg-H2AX

Replicating cells display classes of foci

earlymid

late

granular

lots strong

medium no.

few weak

few strong

blurred

0

5

10

15

20

25

30

35

40

% n

ucle

i

S-Phase substage

gH

2A

X p

att

ern

g-H2AX Foci in replizierenden Zellen (A549)

granular

lots strong

medium no.

few weak

few strong

blurred

H. Scherthan, unpublished

BrdUgH2AXDNA

Minipig skin, Ahmed et al . 2012PLoS One 7:e39521

gH2AX 53BP1DNA PBLs

Apoptotic cells display strong gH2AX fluorescence

Another source of background: granulocyte autofluorescence

gH2AX a.casp.3DNApig skin

Ahmed et al . 2012 PLoS One 7:e39521

g-H2AX Focus assay: IF

n nuclei = 68 (x4,25) 42 (x2,6) 16

Research: Enumerate (manually) foci numbers in 40 positive cells,

or in up to 800 negative cells (Rothkamm & Löbrich 2003; & others)Rapid diagnosis: enumerate (50) - 100 cells (Multibiodose)

gH2AX fpc linearly correlate

with doses < 2Gy (30min pIR, fibroblasts)

Rothkamm and Lobrich 2003 PNAS: ~40 γH2AX foci at 5‘ /1Gy; ~7foci @ 0.2Gy (35DSB/Gy) - fibroblasts

Lobrich 2005 PNAS: ~20 γH2AX foci/lymphocyte

ZIP1

DAPI

DSBs (3’ end labeling)

@ 100Gy

wt SK1

spo11∆ (no eDSBs)

Direct visualization of IR-induced DSBs –lessons from yeast

(3’-end labeling)

1DSB /5 Gy /cell

Illner & Scherthan 2013 PNAS

40DSBs @1Gy in human ≈ 40 DSBs @ 200Gy in yeast nuclei

meiotic chromosomes

Gy

time

Repair foci

DNA

(nuclei)

0h 2h 24h

DNA-Repair Foci in Leukocytes: sensitive, but rapidly declining marker of IR exposure

g-H2AX Foci in in vitro X-IR LCLs

5

10

15

20

25

30

35

40

0,2

5

48

h

fo

ci /

nucl

eus

1 Gy

1 Gy

1 Gy

0,2 Gy

0,2 Gy

0,2 Gy

0 Gy

0 Gy

0 Gy

0,25 2 5 8h 24h 48h

Scherthan et al. 2007 RadRes

0

0

5

10

15

20

25

30

35

40

15' 2h 3,5h 5h 8h 24h

av. fo

ci/

nu

cle

us

gH2AX foci in LCLs post 1Gy

1Gy AT15

1Gy AT17

1Gy P195

1Gy 96P473

1Gy P0040 h

1Gy P0774 h

1 Gy Ko1

1 Gy Ko2

1 Gy Ko3

0Gy Ko1-3

Genetic background influences RIF formation – effects of NBS1 or ATM

deficiencies

53BP1 foci in LCLs post 1Gy

0

5

10

15

20

25

30

35

15' 2h 3,5h 5h 8h 24h

av. fo

ci/ n

ucl

eus

1Gy AT15

1Gy AT17

1Gy P195

1Gy 96P473

1Gy P0040 h

1Gy P0774 h

1 Gy Ko1

1 Gy Ko2

1 Gy Ko3

MW 0Gy 3

Cell Lines

All cell lines that lack ATM or NBS1 fail to induce the full level (30) of gH2AX or 53BP1 foci 15’ after 1Gy IR. Control

H. Scherthan, unpublished

Pittfalls:

• Tissues with endogenous DNA damage, such as testis, lymph nodes • aging cells • replicating cells

Sedelnikova et al. NCB 2004

Senescent cells contain increasing # of persistent gH2AX foci

Age

of

mic

e m

on

ths

% focus-free cellsav. focus # in +cells

Wéra et al. Rad Res. 2013Menegakis et al. IJRB 2009

The fraction of cells with γH2AX foci 24h after IR correlates with clonogenic survival

ATM-p

53BP1

0

5

10

15

20

25

30

35

A B C D

Investigator in time order at mic.

Fo

ci /

cell

5

8

11

44

46

62

69

82

101

98

107

111

129

135

155

292

362

305

143

215

75

236

218

189

288

Variability in manual analysis

Focus assay: variation among investigators => automation

InstRadBioBw

© InstRadBioBw

Analysis / semi-automated image capture and

processing

• motorized mic

• motorized slide table

• e.g., MetaSystems fluor. imaging sys.

Computer aided focus analysis

• Single cell

analysis

• Data output

• Extended

focus

image

InstRadBioBw

Automated scanning & image analysis – not so

variable?

InstRadBioBw

Reagent-induced variation

different secondary Abs –> variation

neue sek.-Cy5-AKs (Dianova) ; LZ-HS v. 29.04.09 ; verd. 1:200

0

4

8

12

16

20

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

n Foci/Zellkern

Zell

kern

e (

%)

1Gy gamCy5

1Gy magCy5

1Gy bagCy5

1Gy damCy5

neue sek.-Cy5-AKs (Dianova) ; LZ-HS v. 29.04.09 ; verd. 1:200

0

4

8

12

16

20

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

nFoci/Zellkern

Zell

kern

e (

%)

0Gy gamCy5

0Gy magCy5

0Gy bagCy5

0Gy damCy5

Performance - sec. Antibodies Performance - sec. Antibodies

Consequence: standardize your staining protocol, run positive & negative controls

• M. Lassmann, Clinic of Nuclear Medicine, Univ. of Würzburg, GER

In vivo: repair focus formation in PBL after thyroid cancer therapy with I-131

DTC: Lymph node & thyroid rest treatment by ablation w 3,7 GBq I-131

High-riskLow-risk

OP

I-131 Ablation

(3,7 GBq)

Tumor stage: pT1, pT2, pN0, cN0 pT3, pT4, N1, M1

(TNM 6. Aufl.)3 – 4 wkT4 withdrawal or

rhTSH

DTC treatment scheme

Lassmann et al. 2010 J.Nucl. Med.

Eberlein, Lassmann & Scherthan, in prep.

differentiated thyroid tumor

Repair foci estimationPhysical dosimetry

Dosimetry – absorbed dose to the blood

Mean specific absorbed dose to the blood: 0.105±0.067 Gy/GBq (25 Pat.)

Mean absorbed dose to the blood: 0.39±0.40 Gy, range 0.2 – 2 Gy

0

50

100

150

200

250

300

350

0 24 48 72 96 120Zeit nach der Verabreichung in Stunden

Blu

tdo

sis

in m

Gy

blood dose

Mean Dose Rate: @ 2h 15.4±3.2 mGy/h

@ 24h 3.4±0.9 mGy/h

@ 48h 1.1±0.4 mGy/h

@ 72h 0.8±0.3 mGy/h

@96h <0.5 mGy/h

0.0

0.1

1.0

10.0

0 24 48 72 96 120

Stunden nach Applikation

Akti

vit

ät

im B

lut

(% /l)

blood

whole body activity

(% appl. act.)

Lassmann et al. J Nuc Med. 2010

0.0 0.2 0.4 0.6 0.8

0.0

0.2

0.4

0.6

0.8

Nu

mb

er

of E

xcess F

oci / C

ell

(g-H

2A

X)

Number of Excess Foci / Cell (53BP1)

Good correlation of gH2AX & 53BP1

repair foci

Lassmann et al. 2010, J Nucl Med

0.001

0.01

0.1

1

g-H2AX

0 24 48 72 96 120

Time after Administration of I-131 / h

Nu

mb

er

of

Ex

ces

sF

oc

i/

Ce

ll

P21

P4

P6

P7

P8

P9

P10

P11

P12

P14

P15

P16

P17

P18

P19

P20

P22

P23

P24

P25

P26

P553PB1

0 24 48 72 96 120 1440.001

0.01

0.1

1

0.001

0.01

0.1

1

g-H2AXg-H2AX

0 24 48 72 96 120

Time after Administration of I-131 / h

Nu

mb

er

of

Ex

ces

sF

oc

i/

Ce

ll

P21

P4

P6

P7

P8

P9

P10

P11

P12

P14

P15

P16

P17

P18

P19

P20

P22

P23

P24

P25

P26

P5

P21

P4P4

P6

P7

P8

P9

P10

P11

P12

P14

P15

P16

P17

P18

P19

P20

P22

P23

P24

P25

P26

P5P553PB1

0 24 48 72 96 120 144

gH2AX 53BP1

Lassmann et al. 2010, JNM

Focus analysis among DTC patients treated with ~3.5 GBq I-131:high inter-individual variability

y = 0.0089x - 0.1663

R2

= 0.9682

y = 0.0093x + 0.0731

R2

= 0.8701

y = 0.0202x + 0.1401

R2

= 0.7578

0

0.1

0.2

0.3

0.4

0.5

0 5 10 15 20 25 30 35 40 45

4 hr Absorbed Dose Increment [mGy]

Nu

mb

erof

Ex

cess

Fo

ci /

Cel

l

P5

P4

P21

Linear (P21)

Linear (P5)

Linear (P4)

Correlation foci / physical dosimetry

RIT: increased foci numbers at low dose rate

Hours after I-131 administration

0.01

0.1

1

10P4 P5

0 24 48 72 96 120 0 24 48 72 96 120 0 24 48 72 96 120

P14 P19

0.01

0.1

1

10

0 24 48 72 96 120 1440 24 48 72 960 24 48 72 96

P18

0.01

0.1

1

10P23 P24

0 24 48 72 96 0 24 48 72 96 0 24 48 72 96

P25

P7

Hours after I-131 administration

0.01

0.1

1

10P4 P5

0 24 48 72 96 120 0 24 48 72 96 120 0 24 48 72 96 120

P14 P19

0.01

0.1

1

10

0 24 48 72 96 120 1440 24 48 72 960 24 48 72 96

P18

0.01

0.1

1

10P23 P24

0 24 48 72 96 0 24 48 72 96 0 24 48 72 96

P25

P7

Induced foci relative to absorbed dose rate

increases with w decreasing dose rateiF

oci/

mG

y /

h

00

00

00

00

00

01

0 h 2 h 48 h 72 h 96 h

gH

2A

X f

oci /c

ell

gH2AX Foci post RIT (~3,7MBq 131-I)

P5

P6

P7

P8

P9

Dosisrate

Lassmann, Scherthan, et al. 2010; J Nuc Med

Calibration of the Focus assay for radionuclide incorporation

• Monte Carlo simulation of decay and dose built up according to the volume & geometry of the vials to realize doses to the blood (0-95mGy)

► 3 healthy individuals; 7 experiments: with I-131 (4) and with Lu-177 (2+1)

► Activity in I-131/Lu-177 aliquot measured by germanium detector

► Blood samples (3,5ml) partitioned

to different tubes + 1ml NaCl diluted radioactive solution

► Incubation for 1h at 37°C under mixing

► Sample preparation as published(Lassmann et al. 2010, NucMed)

► Mic analysis of γH2AX+53BP1 FPC

Eberlein et al. 2015, PloSOne

γH2AX 53BP1

Fit inklusive Punkte aus externer Bestrahlung (100mGy-1Gy)

In vitro calibration of fpc yield and absorbed dose to the blood after in solution exposure to 177Lu und 131I radionuclides

(Cooperation w M. Lassmann, Nuclear Medicine WÜ University, DE)

RIF (radiation induced foci): γ-H2AX-foci colocalized w 53BP1 (manual count w DBP-filter)

No nuclide and test person specific behaviour

Eberlein et al. 2015, PloSOne

FPC calibration curve of 1h 131I, 177Lu radionuclide-

treated PBL samples

Eberlein et al. 2015, PloSOne

-1y=0.012  RIF/cell+0.015 RIF/cell mGy x

Partial body, acute high dose g-irradiation: Focus yield in skin & blood cells

**

non-IR 4h post 50Gy

Pig-skin

Agay, et al. Exp. Hemat. 2010

Pig model

InstRadBioBw

CRSSA, F

-

5

10

15

20

25

30

0 h 4 h 20 h 3 d 28 d 49 d 70 d

γ-H2AX

53BP1

overlap.

*

**

**

**

**

0,05

**

av.

# o

f R

IF /

ce

ll

-

20

40

60

80

100

0 h 4 h 20 h 3 d 28 d 49 d 70 d

% of cells w ith foci

% o

f ce

lls w

ith

fo

ci

High dose rate IR - 50Gy γ-IR doesn‘t saturate the DDR

gH2AX 53BP1

20h 3 d 28 d0h 70 d4h

Ahmed et al. PloS One. 2012

**

No correlation of foci # in skin & blood after 50Gy partial body g-irradiation

non-IR 4h post 50Gy

Pig-skin

InstRadBioBw / CRSSA, F

Range 1-3 1-8 1-4 foci/+cell

av. gH2AX foci in pig

PBCs

0,0

0,1

0,2

0,3

0,4

1

av. f

oci/

cell

T0

H4

D25

PB Leukocytes

0h 4h 25d pIR

-

5

10

15

20

25

30

0 h 4 h 20 h 3 d 28 d 49 d 70 d

γ-H2AX

53BP1

overlap.

Ahmed et al. 2012;

Lamkowski et al.

2014, PloSOne.

RIF in 50Gy IR

keratinocytes

-1

19

39

59

79

99

1 2 3 4 5 6 7 8

% c

ells

wit

h ≥

1 f

oci

foci no. / cell

Foci distribution in IR-damaged PBL

control

4h

24h

168h

-6

-4

-2

0

2

4

6

8

10

12

14

u

Dev. from Poisson distr. (u)

indicates PBI

4h 24h 168h / 4h X in vitro

High dose rate partial body γ-IR doesn‘t saturate the DDR

Lamkowski et al. 2014 PlosOne

100

80

60

40

20

0%

1%

2%

3%

4%

5%

6%

control 4 h 24 h 168 h

% P

BL

w p

an-γ

H2

AX

h post IR

γH2AX 53BP1

**

** P=0.0029

High dose & dose rate partial body γ-IR: Appearance of pan-yH2AX nuclei indicates PBI

early after the exposure

Lamkowski et al. 2014 PloSOne

How many breaks/focus?

Error prone repair in foci

@ high doses?

Question

High doses of IR induce more DSBs than Foci

Growing # of DSBs / Repair Foci with Dose probability of misrepair

max. induced DSBs vs. induced foci in lymphocyte

nuclei

-5

5

15

25

35

45

0 1 2 3 D (Gy)

av.

foci

/ cell

theor. DSBs

#1 0,5h

#1 2h

#2 0,5h

#2 2h

*

Ward 1991

Scherthan et al. 2008

DSBs

ind.foci

*

Repair

IR w high doses /dose rates

correct

# of Chromosome aberrations

Measure for misrepair

erroneous

mFISH analysis: translocation yield in lymphocytes

24 human chromosomes in different colors translocations rates

as a measure of misrepair

Minimal Number of (misrepaired) Breaks (MNB) for this cell: 3

Scherthan et al. 2008, BBRC

(±): standard error of the mean. a based on the 30 min value; MNB from Sky data.

Dose (Gy)

Foci #0,5 hpost IR

2 hMNB /

induced focusa

maxim.

break #

/ focus

0.5 4.15 ±0.4 4.07 ±0.4 0.072 4,8

1 9.31 ±1.1 5.84 ±1.1 0.070 4,3

3 16.8 ±2.3 10.8 ±1.9 0.26 7,1

High doses of X irradiation cause accumulation of DSBs in repair foci increased translocation yield (misrepair)

Scherthan et al. 2008, BBRC

Conclusions

Not always a linear dose relationship

high inter-individual variation

DSB No. / focus increases with dose

source of mutation and complex exchanges.

Low dose effects

Conforth 2006

DNA Repair occurs in foci (”factories”)

Conclusions

DSB Repair (γH2AX) Foci

- High sensitivity

- High inter-individual variability. Rapid decline

- Residual damage (>24h) can correlate with radiation sensitivity. In skin indicates IR for weeks

- Good indicator of WB exposure, dose reconstruction difficult

- Problematic for dose reconstruction after partial body exposure

People involved

Inst. für Radiobiologie BwHarry Scherthan

Gerrit Schrock

Michael Peper

Emad A. Ahmed

- Helmholtz Zentrum München (mFISH)

Ludwig Hieber, Herbert Braselmann,

Horst Zitzelsberger

- Nuclear Medicine Univ. Würzburg

Michael Laßmann

Uta Eberlein

IRBA, Bretigny sur Orge, F (minipigs)

Michel Drouet

Fabien Forcheron