WORKSHOPWORKSHOP“RADIAZIONI IONIZZANTI: NUOVI MODELLI PER LA STIMA DEL RISCHIO”

1 ottobre 2008 , ENEA, Roma

OncogenicOncogenic bystander radiation effects in bystander radiation effects in PatchedPatched heterozygous mouse cerebellumheterozygous mouse cerebellum

Mariateresa MancusoBAS-BIOTECMEDENEA CR-Casaccia

Mouse modelMouse model

Gorlin Syndrome

PTCH protein Shh pathway

Hypersensitivity to ionizing radiation-induced tumorigenesis

Ptc1 Ptc1 neo6neo6--7/+7/+

PTCH

SMO

IR-induced tumorigenesis in brain and skin

Basal cell carcinoma

1. Develops only in irradiated mice (10- 20% )

2. Can be induced by irradiation of newborn and adult mice

3. Late onset (45 weeks)

Mancuso et al., Cancer Res, 2004, 2006Pazzaglia et al., Cancer Res, 2004

0 20 40 60 80 1000

5

10

15

20

25 P2

P90

Latency (Weeks)

BC

C-li

ke tu

mor

inci

denc

e (%

)

1. Relatively low spontaneous rate (about 7%)

2. Greatly enhanced by irradiation in neonatal age

3. Early onset (20 weeks)

Pazzaglia et al., Oncogene, 2002, 2006a, 2006b

Medulloblastoma

0 10 20 30 40 50 600

25

50

75

100P2P4

P1

0 Gy

Latency (Weeks)

Med

ullo

blat

oma

inci

denc

e (%

)

Irradiation set-up for neonatal mice

In-vivo bystander effect?

Shield: lead bar (Ф 3mm) 3 Gy

0 10 20 30 40 500

10

20

30

40

50

60

70

WB 3 Gy (n = 37)

CN (n = 51)

Bar 1 (n = 40)

Bar 2 (n = 45)

Weeks after irradiation

Med

ullo

blas

tom

a in

cide

nce

(%)

Shield: individual hoods(4mm-thick walls)

16 mm

4 mm

Φint = 10 mm

Improved irradiation set-up for neonatal mice

Dosimetry and Monte Carlo simulationDosimetry and Monte Carlo simulation3 3 GyGy x 0.012 = 0.036 x 0.012 = 0.036 GyGy

0 10 20 30 40 500

10

20

30

40

50

60

70

WB 3 Gy (37)SH 3 Gy (46)

WB 0.036 Gy (34)0 Gy (51)

Weeks after irradiation

Med

ullo

blas

tom

a in

cide

nce

(%)

The incidence of medulloblastoma in shielded cerebellum is orders of magnitude higher than can be explained by the

0.036 Gy dose resulting from photon scattering

High medulloblastoma incidence in shielded cerebellum of Ptch1+/- mice

P < 0.0003

P < 0.0011

4016 T TA A CC CG

T4016

4016 T TA AC CG

Biallelic Ptch1 loss (LOH) in tumors from WB or SH irradiated mice

Genetic damage is a critical component of

in-vivo oncogenicbystander responses

Ptc

h1

Ptc

h1

The presumed cells-of-origin of medulloblastoma are undifferentiated precursors of granule neurons that occupy the external granule layer (EGL) of the developing cerebellum

P2

Ruiz i Altaba A. et al. 2002

Induction of DSBs in irradiated and bystander EGL

WB 3 Gy SH 3 Gy

γ-H2AX staining declined in irradiated EGL at later times whilst remaining very low or undetectable in shielded EGL

0.5 h post-irradiation

0 10 20 30 40 50 60 70 800

2

4

6

8

SHWB

0.036 Gy15253545

Time post-irradiation (h)

Num

ber o

f apo

ptot

ic c

ells

(%)

Apoptosis in irradiated and bystander EGL 3h

6h18

h

EGL

ML

WB SH

A steep increase in apoptosis was detected at 6 h in the EGL of shielded mice relative to internal controls (WB 0.036 Gy)

0

5

10

15

20 SH 8.3 GyWB 0.1 Gy

3h 6h4.5h

18h

***

***

No

of a

popt

otic

cel

ls (%

)

0.0

0.5

1.0

1.5WB 0.1 Gy

*

SH 8.3 Gy

4.5h

6h

**

3h 18hN

o of

cel

ls w

ithγ-

H2A

X fo

ci (%

)

Radiation bystander damage at higher dose

SH 8.3 Gy WB 0.1 Gy

DN

A D

SBA

POPT

OSI

S

Short-term cellular responses were not specific ofradiosensitive Ptch1 +/- mice

TPA

Cx43

Nms

COX-2

GJICs are crucial in mediating bystander responses in mouse CNS in vivo

Model of GJICs

0

10

20

30

40

50

***

No

of a

popt

otic

cel

ls (%

)

0

1

2

3

4 SH 8.3 Gy

SH 8.3 Gy + NmsSH 8.3 Gy + TPA

**

No

of c

ells

with

γ-H

2AX

foci

(%)

ConclusionsConclusions

These results represent the first proofThese results represent the first proof--ofof--principle that principle that bystander effects are factual bystander effects are factual in vivoin vivo events with carcinogenic events with carcinogenic potential potential

Genetic damage, mediated by Genetic damage, mediated by GJICs,GJICs, is a critical component is a critical component of of in vivoin vivo oncogeniconcogenic bystander responsesbystander responses

Bystander effects are not specific of Bystander effects are not specific of Ptch1Ptch1--mutant mice and mutant mice and represent a crossrepresent a cross--strain phenomenonstrain phenomenon

1. < 1 Gy

22 mm

4Φint = 10

+ 5 mm

2. 3 Gy

Work in progressWork in progress

0 10 20 30 40 50 60 700

10

20

30

40

50

60

10cGy25cGy50cGy

0 Gy

Med

ullo

blat

oma

inci

denc

e (%

)

Whole-body irradiation

Mariateresa MancusoEmanuela PasqualiSimona LeonardiMirella TanoriSimonetta RebessiVincenzo Di MajoSimonetta PazzagliaMaria Pia ToniMaria PimpinellaVincenzo CovelliOrsio AllegrucciMaurizio Quini

Anna Saran

AcknowledgmentsAcknowledgments

Centro Ricerche Casaccia

Sezione di Tossicologia e Scienze BiomedicheIstituto Nazionale di Metrologia delle Radiazioni Ionizzanti

0 10 20 30 40 50 60 700

10

20

30

40

50

60 SH

10cGy25cGy

50cGy

0 Gy

Med

ullo

blat

oma

inci

denc

e (%

)

0

10

20

30

40

50 **WB 3 GyWB 3 Gy + TPAWB 3 Gy + Nms

No

of a

popt

otic

cel

ls (%

)

Model of GJICs

Cx43

SH 8.3 Gy SH 8.3 Gy + TPA

cx43

β-actin

37 KDa

TPA

Cx43

(200 ng/g b.w., 2-h intervals)

GJICs are crucial in mediating bystander responses in mouse CNS in vivo

Nms

COX-2

(1.5 mg/kg b.w.)

0

10

20

30

40

50

***

No

of a

popt

otic

cel

ls (%

)

0

1

2

3

4 SH 8.3 Gy

SH 8.3 Gy + NmsSH 8.3 Gy + TPA

**

No

of c

ells

with

γ-H

2AX

foci

(%)