Download Recovery response of dividing cells in the thymus of

rNT.J. RADTAT.
BroL.,1976,vol-. 30, No. 5, 409-+17
+3
Recovery response of dividing cells in the thymus of
whole-body gamma -furadiated mic e
D. SUCIV, Z.URAY and M. MANIU
Oncological Institute, 3400 Cluj-Napoca, Romania
(Receiaed14 June 1,976;accepted25 August 1976)
Mice were irradiated with different doses of gamma-rays 30 min after the
administration of s2P-orthophosphate. The dose-response curves determined
at 72 hours after exposure showed an inflection point in the total activity present
in the DNA in thymus and spleen. In the low dose-range, the dose-response
(n:2'5) for the
curves have D6:55 rad (n:2'5) for thyrnus and Do:95rud
spleen. Thirty minutes after the administration of 32P-orthophosphate, the
dividing cells from thymus were partially synchronized by the administration of
80 mg per kg body-weight hydroxyurea. At different time-intervals, the mice
were irradiated with 80 rad, and the total activity of DNA was determined at
A significant maximum of recovery was found
72 hours after synchronization.
at 5 hours (S phase) after the administration of hydroxyurea. In similar
conditions, the dose-response curves corresponding to the Gr, S and M phase
of the division cycle were also determined. The synchronization of dividing
cells induced by hydroxyurea failed in the spleen.
1. Introduction
The recovery of dividing cells from radiation injury has been demonstrated
in different cellular systems (Elkind and Whitmore 1967), including bonemarrow (McCulloch and TilI1962, 196+)and splenic lymphocytes (Okada 1970).
However, in early investigations, it was found that thymic lymphocytes were
unable to recoverfrom radiation damage(Jackson,Christensenand Bistline 1'969,
]ackson and and Christensen 1,972). It was concluded that the mechanism
responsiblefor thymus cell-death is radiation-induced interphase death followed
by cell lysis (Jackson, Christensen and Forkey 1968, Okada t970). More
recently, from dose-fractionation studies, it was suggestedthat the mitoticallyactive cells from thymus can recover from radiation injury (Suciu, Uray and
Abraham 1975). As an extensionof this study, the present work was undertaken
to determine the effect of whole-body irradiation on the dividing cells from
thymus and spleen, previously labelled by the administration of 3zP-orthophosphate. The retention of 32P-DNA in the thymus and spleen was taken as a
measure of both the activity of DNA synthesis and the loss of dividing cells.
The radiation response of dividing cells from thymus and spleen was also
investigated after partial synchrony induced by hydroxyurea, an S-killing and
Gr/S blocking agent (Brown 1975). The results support the assumption that
the mitotically-active cells from thymus and spleen are capable of recovery
from radiation injury without the loss of reproductiveintegrity.
2. Materials and methods
2.1. Radioactiaelabelling
Male mice of the NN{RI strain weighing 28 t2 g were injected intraperitoneally with 25 pCi of 32P-orthophosphate(6 Ci per mmole, IFA
4i0
D. Suciu et al.
Bucharest,Romania) or 25 yCi of 3H-thymidine.(5 Ci per mmole, Amersham,
England).
2.2. Irradiation conditions
Mice were whole-body irradiated in plastic cagesby a 60Cotherapeutic unit
(Theratron 80) (FSD:80 cm, 100 rad per min). The dose-ratewas measured
with a Siemens universal dosimeter. The irradiated and control mice were
given a standard diet.
2.3. DNA determination
Thymus and spleenwere homogenizedin cold 0.1+ N NaCl with a PotterElvehjem homogenizer. The homogenatewas treated with a solution of KOH
to a final concentrationof 0'3 N, and the sampleswere incubated for 24 hours
at 37"C. Perchloric acid was added-to-a final'eoncentration'of 0.2 I{, and the
samples were centrifuged for 10 min at 1400x g. The supernatant fluid ri'as
discarded;and the sedimentwas washed in0'2N HCIO4 and centrifugedfor
10 min at 1400x g. The sedimentwas hydrolysedin 0.2 N HCIO4 for 15 min
at 100'C. Fractions of hydrolysate were used for the diphenylamine colour
reaction (De France and Le Pecq 196l) and the determination of radioactivity.
2.4. Radioaetiztemeasurement
Fractions of hydrolysatewere dried on filter-paper discs, and the s2P-radioactivity was counted with a mica end-window Geiger-Muller counter (Typ.
YAZ) connected to a VA-M-16D
Vakutronik counting system. The
3H-thymidine radioactivity was counted in a liquid scintillation spectrometer
(Betaszint BF-5003), as previously described(Suciu et al. 1975). From these
measurements,the total activity (".p.-. per DNA per organ) and specific
activity (..p.-. per mg DNA) of DNA in thymus and spleenwere determined.
The valuesgiven in figures are means of the results obtained from 8 to 16 mice
( t SEM). Two hours after the administration of 25 p.Ci per mouse
3H-thymidinethe specificactivity was1.6'2+1.1mpcCiper mg DNA in thymus
and 54'0 t 3'6 mpcCiper mg DNA in the spleen. In similar conditions,after the
administration of 3zP-orthophosphatethe specific activity in thymus was
4'l t 0'3 mpcCiper mg DNA and 7'2 t0'7 m1.cCiper mg DNA in the spleen.
These values were obtained after correction of the quenching effect.
3. Results
3.1. DNA labelling
After the administrationof 3H-thymidine or s2P-orthophosphate,
the specific
activity of DNA was determinedat different time-intervalsin thymus and spleen
(figure 1). As has previously been shown (Nygaard and Potter 1959, Pierucci
1967),the incorporation of labelled thymidine into thymus and spleenis almost
complete within 2 hours after injection. In the following 6 days, a continuous
decreaseof the specific activity of DNA can be observedin both thymus and
spleen (figure 1) (Nygaard and Potter 1960, Sanders,Dalrymple and Robinette
196+). It was assumed that reincorporation of labelled thymidine released
through breakdown of cells is negligible (Nygaard and Potter 1960). Thus, the
loss of labelled DNA within the period of severaldays after labelling has been
equatedwith the loss of labelled cells from the tissue(Nygaard and Potter 1960,
Responseofdiz.idingcellstnuhole-ooa|'guff.nlu-.'tL.u.uoww''.U"-
t
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I rso
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N
Q
q
Q
q
f, roa
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t
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\q
S,
tq 5 0
t00
ts
;e
o . f 2 5 4 5 6
DPYS
0 . r 2 5 4 5 6
DNYS
specific activity of DNA in tl
Figure 1. Time-course of the 3H-thymidine
32P-ort
of
.(tl :t^
after admi"it""iit"
in $ 2'3' (o)
analysis *u."p"tfotmed as described
expressed as p
Thannhauser^(19a5) ( x )' Results
14 per cent'
of tnt label' SBV' was less than
admi"i;;;;i;"
after
hours
2
at
values
d'erset al' 1964'Suciu et aI'
Gerber, Gerber, Altman ar
lg75). However, the ass
negligible in thYmu* "1q
of t'ut-tudR-labelled DN
1975
(no* and Prusofi 1965,Myers and Feinendegen
t'p-pNA increasesup to 2 days
32Pinto
:;;"ifi; activity og
oiio continuousincorporationof
in
th"
principl.,
In
1).
(figure
label
\
L
\
R
(J
q
\,,
R
R
s
I
200 300
too
DOSE t nod t
4m
+12
D. Suciu et al.
DNA can be comparedwith the in aitro method of continuouslabelling by the
addition of labelledthymidine into the culture medium (Mak and Till 1963,
Yamadaand Puck 1961). As the purpose of this study is to investigatethe
radiation-recoveryresponseof dividing cells in thymus and spleen,we used the
3zP-orthophosphate-labelling
of DNA as a measureof both the continuous
activity of DNA synthesisand the loss of previously-labelled
cells (figures2,3,
5 and 6). However, further work is necessaryto establishthe mechanismof
continuouslabelling of DNA after the in vioo administrationof 8zP-orthophosphate.
\
s80
Y
N
t 6 0
*R 40
P
N z o
200
400
{ mg/kg
HYDRoYYUREF,
Figure
3. Total activity of DNA
administration
of different
administered 30 min before
percentage of total activity of
sao
600
tooo
.t200
body werghf )
in thymus (o) and spleen (o) at 24 hours after
3zP-orthophosphate was
doses of hydroxyurea.
injection of hydroxyurea.
Results expressed as
DNA at 24 hours after administration of the label.
I
\
N
(, 8 0
.L--r--f\
c
- I
!
t 6 0
s
rT -'k' ' . ,
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$ 4 0
8
01
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a
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EF:FER
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7
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ADHNISTRAflOH
Figure 4. Specific activity of DNA in thymus
intervals after administration of 80 mg per
were killed 30 min after administration of
as percentage of specific activity values
administration of the label.
lt
13
t5
/7
OF HYDROXYUPEN
(o) and spleen ( x ) at different timekg body-weight hydroxyurea. Animals
32P-orthophosphate. Results expressed
of untreated controls at 30 min af,ter
Responseof diz'iding cells in whole-bodygamma-irradiated mice
+t3
I
\ 7 5
N
(,
q
{ 5 4
s
P
N 2 s
2
44
6
8
HOURS AFTER NDHN ISTRNTION
OF HYDROXYUREA
Figure
5. Total activity of DNA in thymus (o) and spleen (o) at 72 hours after
a2P-orthophosphate
administration of 80 mg per kg body-weight hydroxyurea.
was administered 30 min before injection of hydroxyurea.
At differerrt ti-"intervals the animals received 80 rad. Control group was irradiated with 80 rad at
30 min after administration of the label. Results eipressed as percentage of total
activity DNA in the control group.
\
L
i-
R
(,
q
s
P
*
so
";:?
, ::3 ,
'oo
Figure 6. Dose-response curves of the total activity of DNA in thymus. szp-orthophosphate was administered 30 min before injection of 80 -g p", kg body-weight
hydroxyurea. Animals were given different doses of radiation at t hour (.) (dr),
5 hours (o) (S) and 6 hours ( x ) (M) after administration of hydroxyurea. Control
group was unirradiated. Mice were killed 72 hours after administration of
hydroxyurea. Results expressed as percentage of total activity of DNA in the
control group. SEM was less than 8 per cent.
3.2. Dose-response
curoes
Mice were whole-body irradiated with different doses 30 min after the
administrationof 32P-orthophosphate.
The total activity of DNA wasdetermined
in thymus and spleen 72 hours after irradiation. The dose-responsecurves
have a shoulderin the low dose-rangecorrespondingto so and i^ (figure 2).
The inflectionpoint (Elkind and Whitmore 1967)indicatesthat a fractioi of the
thymus and spleenpopulation is representedby cells with an increasedradioresistancein the high dose-range(curve TB and ,SB from figure 2). The
+t+
D. Suciuet al.
parametersof the dose-responsecurves are included in the table. The survival
parameters of spleen cells as determined by other methods ur, 5o from 47 to
78 rad and n from 1'8 to 3'3 (Okada 1970).
rI
I
bs,ralue
(rad)
Tt
Tn
Sl
Ss
Extrapolation
number fr'
f)
3o0t
95
2sot
significance of Ta, ?n, se and ss is given in figure 2 and $ 3.2.
t Determined by extrapolation (figure 2)'
Parametersof the dose-responsecurves shown in figure 2'
3.3. Effect of hydroxyureaon the dioidingcellsfrom thymusand spleen
fft! a.pietion of szP-DNA in thymus and spleenat 24 hours after administration of different doses of hydroxyurea is shown in figure 3. It was
demonstratedthat the S-killing effect of hydroxyureais common for all cell
systems with high rates of cellular proliferation (Sinclair,1965, Philips,
Sternbetg,Schwartz, Cronin, Sodergrenand Vidal 1'967,Whithers 1975). As
the utility of hydroxyureain this investigationis only to induce the synchrony
of dividing celis, we decidedto limit the dose of drug to 80 mg per kg bodyweight (fi-gures+-6). The effect of this dose on the incorporation of
s2P-lorthophosphate
into the thymus and spleen DNA is shown in figure 4.
The pulsl labelling experiment (figure 4) suggeststhe G, block induced by
hydroxyurea,as deierminedby the decreaseof DNA synthesisfew hous after
administrationof the drug.
3.4. Radiosensitizationof diaiding cells in thymus and spleenafter administration
of hydroxyurea
Mice-were given 80 mg per kg body-weight hydroxyurea30 min after the
administrationof 3zP-orthophosphate.At different time-intervalsthe animals
were irradiated with 80 rad (figure 5). The total activity of DNA was
determinedin thymus and spleen^t 72 hours aftersynchronization. If irradiation was carried out at 5 hours after administrationof hydroxyureathe radioactivity of thymus DNA was significantlyincreased(p <0'05) comparedrvith
the vaiue detlrmined for the animals irradiated 6 hours after synchronizationThe f,ecoverymaximum (figure 5) correspondsto the maximum of DNA
synthesis(figure 4).
The ,..orr.ty effectwas absentin the spleen(figure 5). Accordingly' DNA
synthesiswas dlcreasedfor a relativelylong period after the administrationof
(figure 4).
hydroxyurea
curves
The precedingdata allowedthe determinationof the dose-response
szP-orthophosphate
30_minbeforethe
shownin figure 6. The animalsreceived
administration of 80 mg per kg body-weight hydroxyurea. Irradiation was
carriedout at I hour, 5 hoursand 6 hoursafter the partial synchronization. The
gamma-irradiatedmice
of ditiding cellsin whole-body
Response
+15
total activity of thymus DNA was determined72 hours after the administration
of hydroxyurea. As the meancell-cycletime for dividing thymocyteswasfound
to be in the rangeof 6 to 8 hours (Metcalf and Wiadrowski 1966),it is probable
that the time of irradiation correspondsto the Gt (1 hour), S (5 hours) and
G, and M phases(6 hours) of the division cycle. The Gt curve (figure 6) was
exponentiallyrelated to dose, indicating that no recoveryoccurs for this cell
population(Do:115 rad). For the S curvewe found Do:85 rad and n:l'2.
lf irradiationwasperformedat 6 hours(G, and M phase)afterthe administration
of hydroxyurea,about 50 per cent of the total activity of DNA was lost at a dose
of 40 rad.
K
>'
4. Discussion
The data reported in this work are based on the assumption that the
DNA in the thymus and spleenof irradiated
depletionof the previously-labelled
animalscorrespondsto the loss of cellswith labelledDNA (Nygaardand Potter
1960, Sanderset al. 196+,Gerber et al. 1963,Suciu et al. 1975).
The thymic populationis representedby the mitotically-activemedium and
large thymocytesand the non-dividing small thymocytes(Borum 1973,Metcalf
and Wiadrowski 1966). The mean cell-cycletime of dividing thymocyteswas
estimatedto be in the range of 6 to 8 hours. The dividing fraction of cells
representsabout 10 per centfrom the wholelymphoid population. The rnedium
and large thymocytes are continuously transformed into non-dividing small
thymocytes. The processof cell renewalof the small size cells requiresabout
is continuously
3 days(Metcalf and Wiadrowski 1,966). As 3zP-orthophosphate
incorporated(figure 1) but only into the DNA of the dividing fraction of cells
(Ord and Stocken1956),the labellingof DNA in the whole populationrequires
about 2 or 3 days. The incorporationof labelledprecursorsinto the DNA of
thymus cellsby other routesthan cell divisionis improbable(Borum 1973). The
dose-responsecurves determined at 72 hours after irradiation (figure 2 and
table) support the assumptionthat in the low dose-rangethe dividing cellsfrom
thymus are capableof recoveryfrom radiation injury. Due to the short cellcycletime, it is probablethat 72 hours after irradiation,when the processof cell
the remainingfractionof
depletionis terminated(Jacksonand Christensen1,972),
thymic cellsrepresentsurvivingcells(Fabrikant 1972). The experimentsshown
in figure 4 suggestthat the Gt/S blocking effect of 80 mg per kg body-u'eight
hydroxyureais limited to about 3 hours. Then, the DNA synthesisincreases
and the radiation recoveryresponseis clearly evident (figures5 and 6). The
low dose-rangeof the dose-responsecurves (figure 6) correspondingto a
representativefraction of in ztioo synchronizedthymus cells indicates that the
S phaseof the division cycleis the most resistantto radiation. These findings
are in agreementwith previousobservationson the radiosensitivityand recovery
response of other mammalian cell systems after synchrony induced by
hydroxyurea(Brown 1,975).
In the splenic lymphoid population both the large and small size cells are
capableof division (Fliedner,Kesse,Cronkiteand Robertson1964,Hanna 796+,
Post and Hoffman 1970). On the other hand, it was suggestedthat the spleen
lymphoid cells consist of at least two fractions with different levels of radiosensitivity(Nygaard and Potter 1960),Gerber and Altman 1970, Raff 1973,
Streffer 197+),and probable different cell-cycletimes (Fliedner et al. 1,96+.
+r6
D. Suciu et al.
Hanna 1.964, Post and Hoffman L970). It is therefore conceivable that the
attempt to synchronizethe population of dividing cells failed (figures 4 and 5).
The radiation-recovery response of the lymphoid population from spleen is
represented by the low dose-range of the dose-responsecurve reported in
figure 2. It may be assumedthat for both thymus and spleen the high doserange of the dose-response curves (figure 2) reflects the radiation response of
some non-lymphoid cellular systems.
In conclusion,our data support the view that the thymic and splenic dividing
cells are capable of recovery from radiation injury. However, as the recovery
processwas studied almost exclusivelyin cell-survival experiments (Elkind and
Whitmore 1967, Okada 1,970)our results appear as indirect evidences.
'
AcKNowLEDGMENT
We are indebted to Mr. C. Donogany for valuable technical assistance.
Les souris ont 6t6 irradides avec des doses diff6rentes, 30 minutes aprds l'administration
du 32P-orthophosphate. Conform6ment aux analyses faites 72 heures aprds l'exposition,
les courbes dose-r6ponse de l'activit6 totale de I'ADN dans le thymus et la rate pr6sentaient
un point d'inflexion. Dans le domaine des doses basses les courbes dose-rdponse ont
D0:55 rad (n:2,5) pour le thymus et Do:95 rad (n:2,5) pour la rate. Dans une autre
exp6rience, 30 min aprds l'administration du 3zP-orthophosphate, les cellules en division
ont 6tE partiellement synchronis6es par I'administration de 80 mg per kg corps.
A des intervalles de temps diff6rents les souris ont 6t6 irradides avec
hydroxyur6e.
80 rad et 72 heures aprds la synchronisation on a determin6 I'activit6 totale de I'ADN.
On ) trouv6 un maximum de r6tablissement 5 heures (phase S) aprds I'administration de
Dans des conditions similaires, on a d6termin6 aussi les courbes doseI'hydroxyur6e.
r6ponse correspondant d la phase Gr, ,S et M du cycle cellulaire. La synchronisation des
cellules en division par la hydroxyurlert'a pas eu lieu dans la rate.
Miiuse wurden 30 min nach Verabreichung von 3zP-Ortophosphat mit verschiedenen
Dosen bestrahlt. 72 Stunden nach der Bestrahlung konnte festgestellt werden, dass die
Dosis-Effekt-Kurven der DNS-Gesamtaktivitet in Thymus und Milz einen Inflexionspunkt
aufweisen. Im Bereich der niedrigen Dosen zeigen die Dosis-Effekt-Kurven Do:55 rad
In einem anderen Experiment
(n:2,5) fiir Thymus und Do:95 rad (n:2,5) fiir Milz.
wurden 30 min nach der Verabreichung von 82P-Ortophosphat die sich teilenden Zellen
Kcirpergewicht partiell
durch Gaben von 80 mg Hydroxyharnstoff/kg
aus Thymus
synchronisiert. In verschiedenen Zeitabstdnden wurden die Mhuse mit 80 rad bestrahlt
vnd 72 Stunden nach der Synchronisierung die Gesamtaktivitiit der DNS bestimmt.
Ein bedeutsames Erholungsmaximum bei 5 Stunden (Phase S) nach der Verabreichung
von Hydrohyharnstoff wurde gefunden. IJnter gleichartigen Bedingungen wurden auch
In der
die Dosis-Effekt-kurven, fiir die Phasen Gr, S und M des Zellzyklus bestimmt.
Milz fand die Synchronisierung durch Hydroxyharnstoff der sich teilenden Zellen nicht
statt.
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;
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I