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Hethada. Hice. Carworth Fatms No. 1, lO-to-S2-werk-old teamla mice were

ured in these studlea. They were housed, 8 t o a cage, on a bed o f 8terUised

co'm cob., and fed Bock1and coaplete r p k e diet. Winking water, provtded

ad libitum, waa acidified to pH 2.5 with HClt to' control Peeudomnee Infectlone. - * + I

b

Srradiatlon. Irxadiation procedureo were idsnticdt i o tbse pliwiourly

dercrlbed(4). Total-body x-ray, frab a General Blect t ic W t m n , 250 x-ray 4 . I -

machin@ delivering an everage w t p u t of 60 Wmin, vw admlnirter;d t o the ;ice '

vhiah were confined during irradiat ion i n perforated luataroid tuber an a lacite

turntable that rotated a t 3.5 ~h~eical: factore were$ 250 kv, 30 ma, 0.25 uun

Cu p l w 1.0 ~mn Irl f i l t e r ; BVL 1,04 mm Cut target distance 79.5 cm.

"Shielded mice" (850 R L.8.) were placed on the turntsble and had the i r

entire l e f t hiad legs restrained by a rubbar band under a tunnel-shaped, 3 mar lead

rUe ld during the p a r t i d body irradtatlon. 850 B x-ray war delivered to these

ohieldad mice in a ringls doe.. Hfee Vera not aneoehatl8ed during irradiation.

Splenic uptake of 5 9 ~ . The splenic uptake of 59E'e was determined by the

~ e t h o d of Smith(S), ApproxbaPely 0.1 &C of 5 9 ~ e chloride in 0.25 ral. of iootoaic

salfos rolution was injected intraperftonedfy. S i r burs later, spleenrr were

woroved, fired i n Boub's f luid, and their radloactgvity use musured In a w e l l -

type cscbtf l lat ion counter. A starrdard prepared a t the tine of Injection w.s

counted, and splenic uptake of 5 9 ~ e vaa expneaed a* percentage of tha infected

dose,

'Pba rrplenlc uptake of S 9 ~ e wan regarded ae a val id srrimate of erytbro-

poiesir, because those conditions tha t sappreee erythropoluls , like plethora,

decrease uptake of iron; and those that mhance etythropoiesis, l i k e exposure t o

hppblda o r iajecrioa of erythropoiatin, increase the rplenic uptake of radfo-

fran(4). . Moreover, a f t e r r l ag of 24 hkrm, reticulocy toois parallrled the

rplanic uptake of B9~e. -

9

Baaietance t o irradiatfon. Stem c e l l replication was e a t h t e d by noting

I increased reeistance t o irradiation, Shielded mice were given rm I n i t i a l dose of

850 R L.S. Bach day thereafter. a sublethal do- of total-bod y x-ray (100 R,

200 B, or 300 B) waa administered to ir different group of previottrly irradiated,

shielded mice. S i r days a f t e r the total-body irradiat ion, s 9 ~ e Cltg was a w n -

i r te red intrajlerironeally and splenic uptake in 6 hours was aeaeured as deecribed

abow(5). Incraaslng splenic uptake of s 9 ~ e , derpi te expom~tb t o identical doe-

of x-ray., was ipterpreted & widence that stem c e l l replication had occurred,

gioing rise t o more daughter erythrablarts, which incorporated m r e radioiron,

Hppoxia, Hppoxia wae produced by placing mice in a chamber evacuated t o

1 /2 atmosphere f o r 16 hours (overnight) on 3 consecutive nights. The mice were

removed on the morning of the th i rd day. Splenectomy o r eurraguination f o r assay *

of plasma erythropoietin waa carrted out a t tha t time.'

Aesay of plasma erythropoietin. Plasma from groups of 32 experfmental mice

waa pooled and froren Immediately a f t e r bleeding. After thawing a t room tempera-

ture, 1.0 m l of plasma WM injected subnttanuously into a plethoric mmtae prepared

for -say by tha method8 described prevfously(6,7).

Results. Stem cell replication itl the spleen, a8 ahown by increasiag radLo-

resistance, increased markedly during the f i r s t four days a f t e r irradiation, an

interval devoid of erythroid differentiation. Interinittent periods of h$poxia

produced alevaead plasM lev& of dkdogenous erythropoietin, but did not induce

erythropoiesis during the early prol iferat ive ghaf3~ of stem c e l l raplicatlon.

S t e m cell replication. 'Pig, 1 rhom that incremental sublethal dorse of

~ total-body x-ray adainirtered t o mice one day after they had received pa r t i a l body I

I x-ray (850 R L.8 , ) resulted in a correrpandiag dose-response when the splenic up- 1 .

I tab of s 9 ~ a was m u u r a d sir days l a t e r , After day 1, radioreaistance Loreased '

nearly 3 t h a n , raaching r ~ t & k on day 4. After f&l$ng t o a nadir on day 6,

the dore-terpoure di.appaar&, and another peak, higher than thr f i r s t , wm

~tt.UBd 6.y 8.

Platma erythro&etin leva l r , Pig. 2 r h m that plea- from hppoxic mice

5 t o 8 them that of plaema ftem ~ L r r a d f i t e d mice after a e M a r asporure to

i a t e f i r i t t ea t hypo&, P&aoma love18 iLf ahieXdrrd mfee at ambient prssrure did not:

E r ~ t h m p o i e r i 8 ~ P5g. 3 demomtratss that although the rpllentc uptake of

radioiron war a s r e a r e d in normal mice srtpcred to i ia temff tant hppoxS8, there v88

oo increase &t ehi.lded arfce on drys 3 and 5 after 850 R L.S., whethar t h y had

b a a exposed 80 hypoxi. or not, kruover, hppoxia did a o t enhance erythropaiemir

oo day 7, when rplenic uptaka of s9?4 'had Lacreacred .baut %io&d in rhislded mice

endogenow arythropoiatin aLicLFed by hyporia, fa%% re tnduee erythmid d i f fer-

aoriarioa during eh* 5 day8 &mediaraly a f t e r parti.1 body 1rz .dh t ion . Zblr '

m-erte that the e rp thropoi r t i a rargac; cell i r tblporatfily refractory t o the

borumi and nolot char the borrooe is inrotiiated,' $ .~re&tiro' pmpbsa~ t ha t rm

d e An a prsviow prpec(4). The rad iobio logicd method of denwartrating c e t l u l a t

rapliaatfoa eupportr <ronulurfons from aar3ier Prork which rruggested that ~~b~~

hemopo*etic &Cam ceT1 prol5feracfoa, occurred dur- the f i t r t S day8 after p a r t f a l

body X-tag (850 B L, 3.) (4) . Beeaura of the diacrepancier ancouatard when comparing t e a a t r obtained

w f th exogaaw colony-fodng cello (8) .ad sadbgrnour stan c*ll# ( 9 ) , s rrmPaw o f

the ++idace for ear3y etem celL replioarLon l o aecsrsary. Our in i t ia l rork(4)

ahored tha t previous shielding of the left hind lag of mice subaaquently exposed

t o s u p r d e t h a l doses of x-ray effected 100 X euroival. U n l i k e spleens of

I wehieldad mice, which d&ed of bone marrow f a i l u r e follow- total-body x-ray,

apleens of obieldad surviving mice develop& orecroscogia hewpa ie t i c co1onI.rr 4

o r 5 days a f t e r 850 B L, 8. Increasing spLeda upcake of Ibll-labeled iodod-xy-

orld$ne ( 1 8 i ~ - ~ d ~ ) during thoee f irmt 5 days indicated accelerated DNA aynthemis ., . . - 4. ..

There was, however, no evidance of d i f fe ren t lo t ion u n t i l a f t e r day.$, A t ' t h l s

I - time, eplenic uptake of 5 9 ~ e 5ncmaned. and later, ' t he re t iculocyte , leukocyte,

and p l a t e l e t urncantrations began t o -lncr&me. ' T h u , brihg .*the l i r o t 3 days *

.+

a f t e r 850 61 L,S,, tke ogleens of ehieldsd dccs contained c e l l e that were &pable

of forming hemopoietic colonies and rynthcwiaiag DlOA before tb i r different%atioh, '

I n t h i s way preventing death from radiation-induced marrow f615ure(4).

Beod t s reported i n t h i s paper confirm the idea that there is ear ly rep l i - w-

cation of hemopoietic atsm cells! The method i e baeed on the evidence that t'

erythroblasta, which incorporate radloiron, a r i s e from humopoietic rtan ce l l s ( lO, l l ) ,

Other workers have conaidered the mnotmt of radioiron hcotpora ted by e e t h t o -

b las t s , and hence t h e i r number, t o be a function of t he number of stem c e l l s

m a i l a b l e fof erythroid different ie t ion( l2 , lS) . The dbinimhing a b i l i t y a f incre-

mental doaee of radiation t o produce rt maaeurabla les ion har bean uosd aa an

e e t h t e of ce l lu l a r replicatfon(9), When, tharefore, subletbal doses of x-ray

delivered t o colanised epleene of shielded mice progressively f a i l ed to auppreaa

splenic uptdce of 59?e, we concluded that more stem cells were preebnt on each

succeeding day a f f e r 850 B L.8,

The 1cw point of the cumes on day 6 i n Pig, 1, coincides with t he onlret of

~ erythxoid d i f fe ren t ia t ion in the splaem indicated by aplenlc uptake of radio- I Iron(4), Thereadtar the dose-response phenomenon dieappsaro. Preetmtebly, t h i s ir

I

erythropoictin; erythroid.differentietion, as iadicated by aplendc uptake of

radioiron and reticuloaytorim, does not occur u n t i l a f t e r the f i f t h day.

How.doee the replicating stem c e l l tranafonn t o the erythropoietin responsive

I cel l? Rretclxaar postulates that erythropoietin enters the c d l during the 01

phase of the cell cycle(l7). During marrow repopulation, however, most stan cells

are i n the 8 phlee of the aycle, and 01 i e too short t o permit the entry of the

ergthropoietin that would induce differentiation. Bir computer model relates the

duration of Cl a s a fuactlon of the number of cells fn the otem c e l l compartment.

Once t h f s $8 replete, GI lengthem, aud erythroid different iat ion may prowad in

the presence of erythtopoietin. H i r pradictiow(l7) are rtrilriagly sMlar t o

our experimental results. \ . Smmar~ . htowpopulat ion studies using a radiobiological mathod provide

avldence tha t endogenous hemopoietic stem c e l l proliferation in the shielded moure 1

I occurs during the ffrst 5 days a f t e r irradiation, Bxpoeure t o hyporda, suff i- 1 I ~ I c i u r t t o produce elevated levels of endogenous erythropoletin, fa i led to induco

arytbrofd differentiation.durSag the early pr6l iferat ive phase of marrow

repopulation. The data *upport the concept that .the- replication . of .tan &lla, . . 2

per se, renders them refractory t o the fnductios bf e*hroid differeneiation by

and Dr. Eric Sinnnons provided valuable technical aesiatance i n the work. 8 . 4.

4 4

1. Curnay, C. P., Coldwasser, B., Pan, C., 3, Lab, Clin, Mad., 1957, vSO,

534.

. 2. Troup, 8. B., Swfeher, S. I-, Young, L. E., Am. J. &d., 1960, v28,

8

3. lafrha, L. G., Ol&ver, L , Ournay, C. U., Brit. J* B.emNol., 1962,

8. Le.i8, J. 9.. Trobau&, P. E., Jr., Batore, 1964, v2M. 589.

9. T i l l , J. S o , &Cul10&, I. A*, R a 1. A c d . $cia, 1964, ~ll4, 115.

LO. Alp-, 1. L., Cramwre, D., fa The Kfnetica of Cellular Rolitoration,

P. 8. Stohk.n, Jr., rd., Oruna am3 8tratton. 8- ?or&, 1999, 290.

U. DIUI~~~, C. W., Laftb., I. a*. OXioar. R.. Brit. J. Iiaumcol.. 1962,

~ 8 , 461.

1% Bodyon, O., Bebehe, I., Hat. Cancer Inet., Monograph 14, 1964, 169.

14. Suit, 8. D., t.)th., I,. G., Oliver, R., Ellis, I . , Brit. J. E ~ t o l . ,

1957, v3, 165.

U. B t o b l u n , Fa, Jr., Ann. 8. I. A c d . Bci., 19H, v77, 710,

16. B o l d ~ a # ~ r , B e , Conat T 0 p h 8 h D ~ . r . l o ~ n t d BIo~o(R, A* A*

Wecoru, A. k!onmy, edo., &8daial~ Pru8, B w rork, vl, tihap. 7, 1966, 203.

17. f r e t c h r , A. L., Bcianco, 1966, vlS1, 367,

._-_--

I----'- L E G A L N O T I C E 7 1

-

, TNs report as an account of Oovernment sponsored work. Neither the United

S,,,Lea "",. the commlsslon, nor any P ~ R O " ~"i t tng on behalf of the C o m d s d o n :

A: Makes any warranty or representatlon,expreeeed or lmplled, with respect to the accu- I racy completeness, or uaefu[ness of the information conalned in this report,

o r that the use

, ,, .iv tnforma~on, apparatus, method, o r process disclosed In this report not 'niri'J@

. " ~

privately owned rlgbta. or B. any ~ ; ~ t , ~ ~ i t i ~ ~ with respect to the use of. o r for

damages resulllng from the 1 of _ i,,forma~on. amratus, method. o r process disclosed in report.

I in the above, acting on behalf of the commission" Includes any ern-

ployee or conkrctor of ihe commlaalon, or employee nf R I I P . ~ cnntractor. to

extent Ihat

employee or contractor 01 the commlsslon. o r employee of such contractor PreW"'. dlasemhates, or provides access to, any information P J ~ Y M ~ to N s employment o r contract

; the Commisalon, o r his employment with such contractor. I- -- ----- ----- . --

I?XG. 1. Reulatance t o irradiation,. a m a s u n of atem c e l l r e p l i u t i o ~ , 1

was estimated by the splsoic uptake of S g ~ a given 6 dayr a f t e r a sublethal dose

of total-body (TB) x-ray was adminietared t o ahlelded (850 R L. 8. ) mice, Daya

post-irradiation refers t o the day &tar 850 8 L.8, on which the rublethal dore

of r-ta, war admhicttered. BBch point repraa&ta the rnsaa splenic uptake of a

group of at leaat 1 2 Isice, The horfcontal line l o the memn v&ue f o r 53 mice

that recelved no total-body x-ray. The arrow Sadiaatea the adaiaistration of

850 R 1.8. The ordinate i. a lqgarithmic soale.

FIG, 2. Plasma erythropoietia wacr estimated by meamring the erythrocyte

(Rbc) radioiran tncorporation in plethoric asray mfce, Time elapaed a f t e r the

admf.o.irtratlon of 850 R L.9, t o ahieldad d c e fr indicated by npost-lrradiation.t'

Croer-hatohed bare denote mean valuer of pooled plaana from mice exposed t o

intermittent hyporia; open bars depict plasma ac t iv i ty from mice a t ambient

pressura. Brackets enclose * 1 standard er ror of t he mean. Mbers in paren-

tbeaea indicate the number of assay mice.

PIG, 3. Erythropoiasia fn moues aplaens was a t h a t e d by measurin~ the

eplenlc uptake of radioiton. ~tnnbars in paranther- indicate the amber of

elrperhental a a W a from which the mean valoeta (bare) were obtained. Crorr-

hatching, brackets Ad title8 are explained i n the legend idr FIG. 2.

POST- IRRADIAT ION NO X - R A Y

2 0 0 R T.B. 3 0 0 R T.B.

8 5 0 R L.S. (ONLY)

POST- IRRADIATION

POST - IRRADIATION NO X-RAY