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DNA relation in mouse thymocytes labelled with thymidine
Life Sciences Vol. 4, pp. 509-518, 1985 . Pergamon Press Ltd . Great Britain.
vAaIAHILITY IN
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Yousa
THYYCCYTS3 LABSLLSD IIITH TH)!IIIDINS Arthur J. Hale, Fdward H. Cooper and Jeremy D. ifilton Pathology Department, St . Thomas' Hospital Bethnal Sahool, London, 5 .x .1 . sad Yedioal Unit, St . Yary'e Hospital, London, w .2 .
(ßeceived 22 December 196) Leukocytes which are proliferating in a variety of natural and szpsrimental aoaditions show a distinct variation between individual oells in the relation of uptalu of radioactive preoursors of I1ß7A to the amount of DNA synthesised (1,2,j) .
It has been Pound that this individual variation is not
influsnoed by teohnioal autoradiographio or microepsotrophotomstrio variables, the type of prsoursor used, arrest of DNA synthesis, ayaling of synthesis or the general szperimeatal aoaditions but it may bs influsnoed b7 the presence of mature granuloaytea in the samples (j) .
Investigation has revealed that
these mature granuloaytes can degrade thymidine to thymins and dihydrothymins at a oonsiderably faster rate than other leukocytes (4,5,6) thus the variation in uptake of tritiated thymidine (jH-Tdr) into individual leukocytes may be due to aompetion for undegraded thymidine under these ezperimental conditions . Further investigation showed that mouse thymooytes are almost free from this degradative effect (6} . The present study was serried out to see if the usual variation in grain oount~DNA relation existed in mouse thymoaytss which do not degrade thymidine.
* Supported by the flellaoms Trust and the British ~opire Cancer Campaign .
509
510
VARIABILITY IN GRAIN COUNT/DNA RELATION
Vol. 4, No .4
Methods Youss thymocytee prepared from one month old mice, were suspended in Hank's solution with 20~ calf serum (oompliment free) . of 12.5 z 106 tbymooytse/ml were used .
Cell concentrations
They were incubated with 2pC/ml
H-Tdr (s~eoifio activity 1 .9mC/mY) for j0 min, washed, ezpoesd to k{ypotonio conditions (Hank's solution diluted with distilled water 3x1) at 3~ °C for' 10 min, fized (msthaaolsaoetic acid js1), dropped onto chilled elides and stained by the Faulgen method (10 min bydrolysie at 60°C in B HC1) .
The
stained smears were coated with Ilford S5 emulsion ezposed for 24 hr end then dewlopsd .
Photographic maps were prepared, and the grains counted over each
one of a number of cells, the grains removed by treating with Farner'e reducer and the Fsulgsn absorption of each identified nucleus on the cell map measured by an integrating microdensitometer . The degradation of the thymidine was studied by one-directional ahromatograpby on ~hatmsn no .1 paper using ethyl acetate, water, formic acid (60 :30x10) as the eolwnt .
The paper was out up and the radioactivity counted in a
Paokard Triâsrb liquid scintillation epsotroustsr by immersing the paper in a liquid phosphor (6) . Heeults and Disouesixm The variation in grain count (ezpreesed ae a percentage of the mean grain count) with amount of DHA eyntheeiszsd in individual thymocytee ie shown in Fig.1 .
This variation is similar to that found in other proliferating lsuko
cyts systesm.
Ths histogram of the unlabelled and labelled population of calls
in such an ezperiment (Fig .2) shawa that there ie no arrest of sells in the synthetic period (S) or the post-synthetic period (C2 ) as there ie no accumulation of unlabelled cells between the 2c (01) and 4c (02) values nor is there an accumulation of labelled or unlabelled cells at yc (02) . Analysis of the supernatant of the incubation mizture at aero, 15, 45, and 90 min shows that there is virtually no degradation of jfi11'dr to thymine and
Vol. 4, No. 4
VARIABILITY IN GRAIN COUNT/DNA RELATION
511
dihydrothymins during the time of uptake of 3Fi-Tdr by the DNA-synthesising thymooytes (Table) .
zoo
~oo 07 C É
2c
Amount
of
DNA
4c
FIß . 1 . Ths variation in the percentage of mean rain count against DNA for mouse thymocytes incubated with ~-Tdr .
These experiments shop that the variation in grain oount~DNA relatlon found is not due to degradation of 3 H~Pdr .
Thus the similar variation which
was found previously in other proliferating leukocyte systems is probably not due to the thymidine degrading effect of mature granulocytes .
Onoe again it
has been found that this variation is not due to arrest of cells once they have entered synthesis and it is unlikely that this present system differs from the others studied with reference to autoradiographic and microapectrophotometric variables .
512
VARIABILITY IId GRAIN COUNT/DNA RELATION
Vol. 4, No. 4
labelled
~ 10 E Z
2c
Amount
DNA
of
l+~a.
4c
2.
Histogram of the DMA dietribut~en in the labelled and unlabelled populations of oslle to whioh ~Pdr wee made available .
TAHLS Degradation of 3H~hdr by boues Thymooytee in Vitro Time of incubation min
Tdr
T
dihydro-T
02
89 .3 89 .5
9 .7 9.4
15
9~ .6 86 .7
9.4 ~~ .3
2.0
45
87 .6 86 .7
72 .4 10.7
2 .6
90
83 .8 79 .8
~5 .4 77 .6
0.8 2.6
~ .o
~15.0 z 10 6 oslls/ml 2Hadioactive thymine (T) and dibydrothymins (dihydro~P) foz~ed in solution by radiolysis (7} .
Vol. 4, No . 4
VARIABILITY II~T GRAIN COUNT/DNA RELATION
513
Thin variation in grain count~DAA relation is not inconsistent with previous findings of the pattern of DgA synthesis ae found by autoradiography or miarospeatrophotamstry alone (j) .
It is however a reflection of individual
cellular variation in rate of uptake of DOHA precursors and of haw this variation ohangea as synthesis progresses .
It ie thus more likely that this vari-
soon ie a reflsotioa of the rate at whioh individual oells move through eynthesie .
iPe have shown previously that there ie ao cyolic variation in the rate
of synthesis in single oelle.
It is however poeaible that the rate of sya-
thesis in Individual cells shown a syatematia eigmoid variation of different shape for esoh osll or that esah cell shows a oonetant but different rate of DHA synthesis .
It ie also possible that the variation may be due to differ-
ences in degree of utilisation of szogenoue thymidine by oslls progressing at the same egrnthetio rate . Beferenoes 1.
S. H . COOP&E, P. BAH~iAA and A . J . HALE, Brit . J. Hasmat . ~, 101 (196j) .
2.
A. J . HALE and S . H. COOPSH, Acta Haemat . 2~. 257 (1963) .
j.
A. J . HALE, E. H . COOPSH and J . D . YILTOA, Brit . J. Haemat . (in the preen) .
4.
J. C . 1[AESH and S . PSEHY, Arch . Hioohem . Biophye . 101 , 146 (1964) .
5.
J. C . iIAHSH and S. PSEHY, J. olio . Invent . ~ 267 (1964) .
6.
S. H . COOPSH and J. D. YILTOH, Brit . J . Csaoer , (in the preen) .
7.
A. S . SVANS and F. ß. STANFORD, Beture (Load. ) 1~, 762 (1963) .
vAaIAHILITY IN
aaAZx
couNT/DN~ a~.A2Ia~ Ix
Printed in
Yousa
THYYCCYTS3 LABSLLSD IIITH TH)!IIIDINS Arthur J. Hale, Fdward H. Cooper and Jeremy D. ifilton Pathology Department, St . Thomas' Hospital Bethnal Sahool, London, 5 .x .1 . sad Yedioal Unit, St . Yary'e Hospital, London, w .2 .
(ßeceived 22 December 196) Leukocytes which are proliferating in a variety of natural and szpsrimental aoaditions show a distinct variation between individual oells in the relation of uptalu of radioactive preoursors of I1ß7A to the amount of DNA synthesised (1,2,j) .
It has been Pound that this individual variation is not
influsnoed by teohnioal autoradiographio or microepsotrophotomstrio variables, the type of prsoursor used, arrest of DNA synthesis, ayaling of synthesis or the general szperimeatal aoaditions but it may bs influsnoed b7 the presence of mature granuloaytea in the samples (j) .
Investigation has revealed that
these mature granuloaytes can degrade thymidine to thymins and dihydrothymins at a oonsiderably faster rate than other leukocytes (4,5,6) thus the variation in uptake of tritiated thymidine (jH-Tdr) into individual leukocytes may be due to aompetion for undegraded thymidine under these ezperimental conditions . Further investigation showed that mouse thymooytes are almost free from this degradative effect (6} . The present study was serried out to see if the usual variation in grain oount~DNA relation existed in mouse thymoaytss which do not degrade thymidine.
* Supported by the flellaoms Trust and the British ~opire Cancer Campaign .
509
510
VARIABILITY IN GRAIN COUNT/DNA RELATION
Vol. 4, No .4
Methods Youss thymocytee prepared from one month old mice, were suspended in Hank's solution with 20~ calf serum (oompliment free) . of 12.5 z 106 tbymooytse/ml were used .
Cell concentrations
They were incubated with 2pC/ml
H-Tdr (s~eoifio activity 1 .9mC/mY) for j0 min, washed, ezpoesd to k{ypotonio conditions (Hank's solution diluted with distilled water 3x1) at 3~ °C for' 10 min, fized (msthaaolsaoetic acid js1), dropped onto chilled elides and stained by the Faulgen method (10 min bydrolysie at 60°C in B HC1) .
The
stained smears were coated with Ilford S5 emulsion ezposed for 24 hr end then dewlopsd .
Photographic maps were prepared, and the grains counted over each
one of a number of cells, the grains removed by treating with Farner'e reducer and the Fsulgsn absorption of each identified nucleus on the cell map measured by an integrating microdensitometer . The degradation of the thymidine was studied by one-directional ahromatograpby on ~hatmsn no .1 paper using ethyl acetate, water, formic acid (60 :30x10) as the eolwnt .
The paper was out up and the radioactivity counted in a
Paokard Triâsrb liquid scintillation epsotroustsr by immersing the paper in a liquid phosphor (6) . Heeults and Disouesixm The variation in grain count (ezpreesed ae a percentage of the mean grain count) with amount of DHA eyntheeiszsd in individual thymocytee ie shown in Fig.1 .
This variation is similar to that found in other proliferating lsuko
cyts systesm.
Ths histogram of the unlabelled and labelled population of calls
in such an ezperiment (Fig .2) shawa that there ie no arrest of sells in the synthetic period (S) or the post-synthetic period (C2 ) as there ie no accumulation of unlabelled cells between the 2c (01) and 4c (02) values nor is there an accumulation of labelled or unlabelled cells at yc (02) . Analysis of the supernatant of the incubation mizture at aero, 15, 45, and 90 min shows that there is virtually no degradation of jfi11'dr to thymine and
Vol. 4, No. 4
VARIABILITY IN GRAIN COUNT/DNA RELATION
511
dihydrothymins during the time of uptake of 3Fi-Tdr by the DNA-synthesising thymooytes (Table) .
zoo
~oo 07 C É
2c
Amount
of
DNA
4c
FIß . 1 . Ths variation in the percentage of mean rain count against DNA for mouse thymocytes incubated with ~-Tdr .
These experiments shop that the variation in grain oount~DNA relatlon found is not due to degradation of 3 H~Pdr .
Thus the similar variation which
was found previously in other proliferating leukocyte systems is probably not due to the thymidine degrading effect of mature granulocytes .
Onoe again it
has been found that this variation is not due to arrest of cells once they have entered synthesis and it is unlikely that this present system differs from the others studied with reference to autoradiographic and microapectrophotometric variables .
512
VARIABILITY IId GRAIN COUNT/DNA RELATION
Vol. 4, No. 4
labelled
~ 10 E Z
2c
Amount
DNA
of
l+~a.
4c
2.
Histogram of the DMA dietribut~en in the labelled and unlabelled populations of oslle to whioh ~Pdr wee made available .
TAHLS Degradation of 3H~hdr by boues Thymooytee in Vitro Time of incubation min
Tdr
T
dihydro-T
02
89 .3 89 .5
9 .7 9.4
15
9~ .6 86 .7
9.4 ~~ .3
2.0
45
87 .6 86 .7
72 .4 10.7
2 .6
90
83 .8 79 .8
~5 .4 77 .6
0.8 2.6
~ .o
~15.0 z 10 6 oslls/ml 2Hadioactive thymine (T) and dibydrothymins (dihydro~P) foz~ed in solution by radiolysis (7} .
Vol. 4, No . 4
VARIABILITY II~T GRAIN COUNT/DNA RELATION
513
Thin variation in grain count~DAA relation is not inconsistent with previous findings of the pattern of DgA synthesis ae found by autoradiography or miarospeatrophotamstry alone (j) .
It is however a reflection of individual
cellular variation in rate of uptake of DOHA precursors and of haw this variation ohangea as synthesis progresses .
It ie thus more likely that this vari-
soon ie a reflsotioa of the rate at whioh individual oells move through eynthesie .
iPe have shown previously that there ie ao cyolic variation in the rate
of synthesis in single oelle.
It is however poeaible that the rate of sya-
thesis in Individual cells shown a syatematia eigmoid variation of different shape for esoh osll or that esah cell shows a oonetant but different rate of DHA synthesis .
It ie also possible that the variation may be due to differ-
ences in degree of utilisation of szogenoue thymidine by oslls progressing at the same egrnthetio rate . Beferenoes 1.
S. H . COOP&E, P. BAH~iAA and A . J . HALE, Brit . J. Hasmat . ~, 101 (196j) .
2.
A. J . HALE and S . H. COOPSH, Acta Haemat . 2~. 257 (1963) .
j.
A. J . HALE, E. H . COOPSH and J . D . YILTOA, Brit . J. Haemat . (in the preen) .
4.
J. C . 1[AESH and S . PSEHY, Arch . Hioohem . Biophye . 101 , 146 (1964) .
5.
J. C . iIAHSH and S. PSEHY, J. olio . Invent . ~ 267 (1964) .
6.
S. H . COOPSH and J. D. YILTOH, Brit . J . Csaoer , (in the preen) .
7.
A. S . SVANS and F. ß. STANFORD, Beture (Load. ) 1~, 762 (1963) .