Stem cells, radiation response, tissue kinetics and carcinogenesisOhtsura Niwa, NIRS, Chiba, JapanOhtsura Niwa, NIRS, Chiba, Japan

Current ICRP carcinogenesis model and its limitationCellular aspect based model combined withepidemiological data

Target cells not definedTarget cells not definedNeed considering tissue aspect of target cells

Tissue aspect may improve the model fordose rate independence of the linear termage dependent sensitivity to carcinogenesisage dependent sensitivity to carcinogenesisrisk of in utero exposures

mechanistic insight of tissue specific sensitivityg p yand more

ICRP Risk model

LNT i t t d f l t l f di ti t tiLNT: an important and useful tool of radiation protectionRisk = dose x risk value x DDREF x WR x WT

dose： measurements + estimationsdose： measurements + estimationsrisk value: cross population risk transferDDREF: varies with biological endpointsg pWR: varies with biological endpointsWT: rounded-up values

Much of the data are from A bomb survivorsThose in blue all have uncertaintyU i i l l l f L d /dUncertainty particularly large for Low dose/dose rate

The model is cellular process basedpThe data comes from human epidemiology on whole body

Limitation of the cellular aspect based modelas shown in the DDREF issue

D βD2

as shown in the DDREF issueer

risk

high dose rate = αD + βD2

DDREF = αD + βD2

αD

= 1 + β/α D

ed c

ance

g dose a e α β 1 β/α D

DDREF equation assumes

tion

rela

te The linear term is dose rate independent. This comes from the

Rad

iat

low dose rate = αD

This comes from the consideration on the cellular repair process

dose

A study of simple chromosome exchanges violate the rule

High dose rate H l i thHigh dose rateα = 0.18 +/- 0.04β = 0.05 +/- 0.02

How can we explain the dose rate sensitive

linear term, even for the cell based effect ?

Low dose rate

cell based effect ?

Whole body can have Low dose rateα = 0.06 +/- 0.01β = 0

more exceptions

Cornforth, DNA Repair, 5,1182. 2006

The target: stem and progenitor cells in a tissue

stem cells apoptosisprogenitor cells

transit amplifying p p

niche

functional cellsterminally differentiated

Blood circulation RepopulationBlood circulation p pWound healing

W h ld k f ll l f llWe should know more of cellular aspect of stem cellsWe should know tissue aspect of stem cells

Major features of tissue stem cells and progenitors

stem cells progenitorsniche required, for stemness Take hold of niche q

and for protection sometimeresidence life-long residence limited residencedivision capacity large limiteddivision status dormant, but not always rapiddivision type asymmetric and

symmetricsymmetric

DNA li i i l d d l l li iDNA replication immortal strand model controversial

regular replication

specific genes variable by tissues and by stagesspecific genes variable by tissues,side population yes, but not always no

and by stages

Hematopoietic stem cell niche

Sinusoidal endothelial cells

Non specialized osteoblastsSpecialized N cadherin+ osteoblasts

Arai and Suda. Ann N Y Acad Sci. 1106, 41-53, 2007

Non specialized osteoblastsSpecialized N-cadherin+ osteoblasts

Niche is required for dormancy and protection

Stem cells< 1%

Divisioncapacity

O2conc. capacity

Differentiatedll

< 1%

Nature 434, 843, 2005

cells

Hierarchical organization of hematopoietic stem cells?

Asymmetric division and asymmetric replication in stem cells- Cairns’ hypothesis (Nature 1975) -stem chromosomeste

transitDNA chromosome

OK

No

No

immortalll

mortalll No random segregationNo SCE allowed

No

cell cell No random segregationNo SCE allowed

No definite proof, disproved for hematopoietic and hair follicle stem cells

Side population phenotype

on/1

06ce

lls

blue

Ho33342

lony

form

atio

Hoc

hest

5 & 10 min

BM & thymus

co

Hochest redDays in culture

FACS analysis 7 day colony formation35 day colony formation

Low Ho33342 cellsw/ high colony formation

Parmar et al. PNAS 104, 5431, 2007

w/ high colony formation

Powerful lineage tagging method for stem cell studyER-Cre knock-in alleleER Cre knock in allele

ER Cre

Cre + T if +Lac tagged cells

Stem specific promoter

Promoter +Cre + Tamoxifen +

Cre to nucleus

loxPselectable gene

stop lacZ geneselectable gene

lacZ gene

LacZ reporter transgene

loxP

general promoter

stop

general promoter

LacZ reporter transgene Use of a cell specific gene promoter to tag the cells

+4 stem cells tagged by Bmi

Two distinct mouse intestinal stem cells identified+4 stem cells tagged by Bmi

promoter driven Cre Sangiorgio & Capecchi Nat Genet in press

EnterocytesGobletEndocrine

+4 stem

P t C t b l

Crypt base columnar stem cellstagged by Lgr5 promoter driven Cre

Barker et al. Nature 449, 1003, 2007

Panet Crypt base columnar

stem cells

Reversibility of progenitors in mouse testiscommitted spermatogoniastem cells

niche

committed spermatogonia

Lineage tagging of mouse testicular spermatogoniag gg g p g

Buslfan treatmentBuslfan treatment

Nakagawa et al. Developmental Cell, 2007

Many cellular aspect to be studied on tissue stem cellsγH2AXPI

3 Gy to 8 hrembryos

H2AX phosphorylation and p21 activation only after 3 day embryosembryos y y

Many weird damage3 Gy to 3 day

embryos

Many weird damage response are wailting to be discovered in tissue t ll

Day 1.5 Day 2.5 Day 3.5

stem cells

p21β-actin

Tissue aspects of stem cells

Potten & Wilson, in “Principles of Tissue Engineering”, 2007

Long lineage tissues: progenitors by themselves are the targetShort lineage tissues: stem and reverted progenitors are the target

Tissue aspect and possible decline of relative riskb ti iby time since exposure

entire lifeTissue specific turnover time

Target of radiation carcinogenesisIn the case of stem cells, dose can accumulate infinitely,In the case of stem cells, dose can accumulate infinitely,

unless stem compartment has a hierarchyIn the case of progenitors, dose can accumulate

only for a limited duration of the turnover timeonly for a limited duration of the turnover time

The rate of relative risk decline

120000 100000

expected from the Armitage Doll model

When radiation

80000

100000

1000

10000

ence

2

Incidence byage related accumulation

When radiation gives one hit

40000

60000

10

100inci

de

11

2of mutation

1 = α (age)52 = (age)4

0

20000

0 2 4 6 8 10 121

10

1 10

2 = α (age)4

number of hit or age

Relative risk = line 2/ line 1Relative risk line 2/ line 1 Relative risk declines at 1/age

Relative risk declines in the LSS population

1.5

40 - 9

k pe

r Sv

ERRD = αD ERRAA = β/(age)1.7

1.0

ativ

e R

isk

2

3

10 - 19s R

elat

ive

Ris

k

0.5

Exce

ss R

ela

1

40+

20 - 39Exce

ss

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.50.0

Weighted Colon Dose (Sv)

30 40 50 60 70 80 900

40

Age at Diagnosis

Linear increase means 1 hit The real decline rate = 1/(age)2

Pierce & Vaeth, Biostatistics 4, 231, 2003

howeverLinear increase means 1 hitERR should decline by 1/age

The real decline rate 1/(age)loss of initiated cells?

Lung cancer among uranium minors

M3 model fitting to lung cancer SMR of French and Czech minors

0% f

9

8

7ERR decreases 50% for each10-year increase in time since exposure

ER

R

6

5

44

3

2

ERR = (β W + β W ) exp [ (AE 30) + θ(TE 20)]

BEIRVI

Age 30 40 50 60 70 80

1

0

Tomasek et al.R d R 2008ERR = (βMWM + βEWE) exp [α(AE – 30) + θ(TE – 20)]

ERR decreases 50% for Th d li t 1/( )3Rough calculation

Rad Res 2008

ERR decreases 50% for each 10-year increase in time since exposure

The decline rate = 1/(age)3loss of initiated cells?

Rough calculationfor age 40 to 50

Loss of initiated cells? An example of rat mammary cancerAn example of rat mammary cancer

Neutron Tumor per rat Tumor per rat Tumor per ratdose

pNo prolactin

pProlactin immediately

pProlactin 12 mo. later

0 rad 2/24(8 3%)

0/14(0%)

-(8.3%) (0%)

4.1 rad 1/16(6.5%)

6/16(38%)

4/15(27%)

7.3 rad 0/16(0%)

5/15(33%)

3/15(20%)

16 6 0/16 9/17 4/1516.6 rad

0/16(0%)

9/17(53)

4/15(27%)

total 1/48(2%)

20/48(42%)

11/45(24%)(2%) (42%) (24%)

Kamiya et al. Jpn J Cancer Res. 76, 449, 1985

Loss of pre-neoplastic lesions after PhIP treatmentC

olon

Aberrant crypt foci (ACF) induced by PhIP

567

rypt

Foc

i/C

234

Abe

rran

t C

01

0 20 40 60 80

No.

A

R t ( k)

PhIP treatment (3 – 10 weeks)

Rat age (week)

Tsukamoto et al. Jpn J Cancer Res 90, 720, 1999

Tissue aspect and the risk of fetal exposures

13 mothers

Ohtaki et al. Rad. Res. 161, 373, 2004150 in utero survivors

181 survivors

Thirteen pairs of the mother and the child

1st trimester

mothers

2nd trimester

3rd trimester

children

Ohtaki et al. Rad. Res. 161, 373, 2004

Hematopoietic stem cells from fetal to perinatal transition

progenitor cellsstem cells

progenitor cellstransit amplifying

Unstable

functional cells

Unstablestem

terminally differentiated

nicheBlood circulation

Stable stem

Bone marrow niche is established after birthStem cells can be stable only in the niche

Precancerous cells in fetus are rapidly eliminated during postnatal development?

Rapid decline of childhood cancer incidence by year after birth

ALL incidence rate and age in Japan Neuroblastoma in situ in a necropsy case

B k ith & P i 1963

ALL translocation carrier: 1/100 newborn 1/10,000 with ALLSilent neuroblastoma carrier: 1/100 newborn 1/10 000 with NB

Nakamura Rad Res 163, 2005 Beckwith & Perrin, 1963

Silent neuroblastoma carrier: 1/100 newborn 1/10,000 with NB

Elimination by tissue turnover, apoptosis and terminal differentiation

Risk of in utero and childhood exposures

Follow-up of 1958 -1999 on solid tumors2,452 in utero exposed： ERR = 1.0 /Sv 15,388 childhood exposed：ERR = 1.7 /Sv

JNCI 100, 428-436, 2008

OSCC d th t l t diOSCC and other case control studiesleukemia : RR = 1.49/10 mGysolid tumors : RR = 1 45/10 mGysolid tumors : RR 1.45/10 mGyall malignancies : RR = 1.47/10 mGy

ERR of in utero exposure = 50/Gyp y

Brit J Radiol 70, 130-139, 1997

Induction of cancer by fetal and perinatal irradiation in mice

3.8 Gy30) 50 30

ovary

li t l h

liverpituitary20

ncre

ase

(%)

30

40

50

20

30

malignant lymphomabone

myeloid leukemia lung0 10 20 30 40

0

10

Net

in

0 10 20 30 400

10

20

0 10 20 30 400

10

Sasaki J Radiat Res Suppl. 2, 73-85 (1991)17-d fetus

0 10 20 30 40Age at exｑposure (days)

0 10 20 30 40Age at exposure (days)

0 10 20 30 40Age at exposure (days)

Not many tissues exhibit high risk by in utero exposureSensitivity related to the establishment of tissue nicheSensitivity related to the establishment of tissue niche

Stem cell kinetics and radiation risk- A case of Min mouse model -A case of Min mouse model

Tumor number

Small intestine Large intestine

Tumor number

200

250 1 Gy2 Gy

16

20 1 Gy2 Gy

Small intestine Large intestine

100

150

8

12

50

100

4

8

02 10 24 42 48

02 10 24 42 48

Age of mice at irradiation

no IR no IR

Age of mice at irradiation

M. Okamoto and H.YonekawaJ Radiat. Res. 46, 83-91 (2005)No induction after certain ages

Radiation sensitive window coinside withstem cells division

S BB B

Ad lt t t t t t f ft bi th

Tsukamoto

Adult type crypts start to form after birthBifurcation of crypts is in its peak on 12 days after birth

Stem cell radiobiology and the future of the radiation carcinogenesis modelthe radiation carcinogenesis model

stem cells apoptosisprogenitor cells

transit amplifying

f ti l ll

niche

functional cellsterminally differentiated

Importance of stem cell researchCellular aspect of stem cellsTissue aspect of stem cells