A C T A U N I V E R S I T A T I S L O D Z I E N S I S FOLIA BIOCHIMICA ET BIOPHYSICA 2 , 1983
B ł a ż e j R ó z g a , E l ż b i e t a Poz iomska, A neta K oc e va -C h yła DEGRADATION OP 3n_TdR LABELLED DNA OP HUMAN LYMPHOCYTES
DURING STORAGE
I n f l u e n c e o f 3H -thym idine i n c o r p o r a t e d i n t o DNA o f human p e r i p h e r a l b lo o d l ym ph ooytes on damage to lym ph ocyte DNA and p o a s i b i l i t i e s o f s t o r a g e o f 3 H - l a b e l l e d c u l t u r e o f human ly m p h oc y tes i n PBS ( b u f f e r e d p h y s i o l o g i c a l s a l i n e , pH 7 . 2 ) a t 4°C was s t u d i e d by e s t i m a t i o n s o f m o l e c u la r weight o f DNA i n a l k a l i n e s u c r o s e d e n s i t y g r a d i e n t and d e te r m i n a -t i o n o f -th e amoun-t o f v i a b l e c e l l s . The r e s u l t s o b t a i n e d i n e s t i m a t i o n o f m o l e c u l a r w ei gh t o f DNA i n d i c a t e d t h a t s t o r a g e o f 3H-thymidine l a b e l l e d human ly m p h oc y tes c a u s e s DNA d e g r a d a t i o n . The s i m u l t a n e o u s l a c k o f e s s e n t i a l chan -g e s i n ly m p h oc y tes s u r v i v a l s u -g -g e s t t h a t d u r i n -g the s t o r a - -g e -i n d u c e d damage th e DNA damage p r e c e e d e s ly m p h oc y tes
c e l l d e a t h .
I n t r o d u c t i o n
I t h a s been shown i n many s t u d i e s o f mammalian c e l l s and b a c t e r i a t h a t r a d i o i s o t o p e s c a u s e l e t h a l i t y and s t r a n d b r e a k s when i n c o r p o r a t e d i n t o DNA. The 1^C-decay i n d u c e s p o t e n t i a l l y l e t h a l l e s s i o n s i n b a c t e r i a l DNA [ 17 ]. ^ " ’ i o d i n e i n c o r p o r a t e d i n t o DNA a s th e thymine a n a l o g u e 5 - / I / - i o d o d e o x y u r i d i n e c a u s e y l e t h a l i t y , m u t a t io n s and d o u b l e - s t r a n d b r e a k s i n E. c o l i [ 1 5 ] » i n p ha ge s T 1 and T^ [1 9 , 2 0 , 2 9 ] , and d e c r e a s e s s u r v i v a l o f ma-mmalian c e l l s [ 2 , 7 ] . B o y d e t a l . [ 1 ] h ave shown t h a t t h e r a -p e u ti c d o s e s o f 1 2 5 i a r e c a p a b l e o f p ro d u c in g chromosome d a-mage i n human p e r i p h e r a l ly m p h o c y t e s. The l e t h a l and m uta g eni c e f f e c t s o f ^2 P i n c o r p o r a t e d i n t o b i o l o g i c a l sy st e m s have been a l s o w e l l dokumented [13» 14 , 16, 2 2 ] ,
3
i a a t o x i c a g e n t when i n c o r p o r a t e d i n t o DNA o f mammalian o e l l a , t o o . I t ha a been e a t i m a t e d t h a t th e e f f e c t o f t r i t i u m a r e a i m i l a r to e q u i v a l e n t d o se a o f X or g a m m a -r a d i a t io n [ 6 , 9 , 2 8 ] , T r i t i u m i n c o r p o r a t e d i n t o Sa c c h a ro m y c es c e r e v i a a e in d u c e a r e p r o d u c t i v e d e a t h , g en e c o n v e ra io n a and m u t a ti on a a i m i l a r to th o ae pr od uc ed by an e q u i v a l e n t doae o f g a m i n a -r a d ia ti on [ 4 ] . H-Thymidine can prod uce chromoaome a b e r r a t i o n a when add ed to- c u l t u r e s o f human lym ph ocytea i n v i t r o [ 2 3 ] . % - T r i t i u m dec ay c a u a e s a i n g l e - a t r a n d b re a k a i n C h i ne s e h am ster c e l l s V 7 9 [ 3 ] . D a ta on i n f l u e n c e o f % - t h y m i d i n e i n c o r p o r a t e d i n t o DNA o f human p e r i p h e r a l b lo o d lym- p h oo yte s on th e damage to DNA a s w e l l a s p o s s i b i l i t i e s o f s t o r a g e o f l a b e l l e d ly m p h oc yte s a r e l a c k i n g . We have been a t t e m p t i n g to c h ec k whether th e p r o c e s s o f s t o r a g e o f ^ H -t h y m i d in e - la b el l e d hu-man lym p hoc ytea i n a o l u t i o n o f PBS ( p h o s p h a t e - b u f f e r e d s a l i n e ) a t 4°C i n f l u e n c e s th e DNA d e g r a d a t i o n and c e l l s u r v i v a l . PBS was u s e d aa a t o r a g e medium s i n c e PBS t re a tm e n t d oe s n ot prod uce t o x i -c i t y nor produ oe d e g r a d a ti o n [ 3 3 ] .
M a t e r i a l e'id methoda
The lym ph ocyte c e l l s u a e d i n th e a e e x p e r im e n t s were i s o l a t e d from h e p a r i n i z e d b lo o d by th e s e d i m e n ta t i o n method i n v o l v i n g CM- - c e l l u l o s e . G r a n u l o c y te s and m ac roph ag es were removed owing to t h e i r a d h e s i o n to g l a a s a t 3 7 °C . The ly m ph o c y tes were c u l t u r e d i n a 199 medium c o n t a i n i n g 10% c a l f serum, a n t i b i o t i c and p h yto-h e m a g g l u t i n in a t 37°C f o r 48 yto-h . Tyto-he lympyto-hocy te DNA was l a b e l l e d w ith % - t h y m id in e w ith s p e c i f i c a o t i v i t y 5 mCi/mmol f o r 24 h , a t a f i n a l a c t i v i t y o f 2 p C i / m l . The c e l l s u r v i v a l was d e ter m in e d by Trypan B l ue s t a i n i n g and l a b e l l i n g '’ "'cr. M ol e c u la r w e ig h t o f DNA w a 3 e s t i m a t e d by u l t r a c e n t r i f u g a t i o n i n a l k a l i n e a u c r o a e d e n s i t y g r a d i e n t a c c o r d i n g to M c G r a t h a nd W i l l i a m s [ 2 4 ] . 5 -2 0# l i n e a r s u c r o s e d e n s i t y g r a d i e n t s i n 0 .1 M NaOH, 0 , 9 M NaCl and 1 mM LDTA were u a e d . The c e l l s were l y s e d d i r e c t l y on th e to p o f th e g r a d i e n t f o r 30 m in u te s a t room t e m p er a tu re ( t h e l y s i n g a o l u t i o n c o n t a i n e d 0 .5 M NaOH and 0 .1 M EDTA), and were c e n t r i f u g e d a t 35 000 rpm i n a Beckman SW 60 r o t o r f o r 2 h a t
1 0 °C . The. g r a d i e n t s were c a l i b r a t e d w ith th e phage T^DNA ( 7 3 S ) . The a v e r a g e m o le c u l a r w ei g h t o f DNA was e s t i m a t e d from the S t u - d i e r ’ s e q u a t i o n [ 3 1 ] .
Re s u l t s
T y p i c a l s e d i m e n t a t i o n p r o f i l e s o f % - l a b e l l e d DNA from hyman lym ph o cy tes i n 5 -2 0 ^ a l k a l i n e s u c r o s e d e n s i t y g r a d i e n t a f t e r d i f f e r e n t ti m e s o f s t o r a g e o f ly m p h oc y tes a r e shown i n P i g . 1.
The v a l u e s o f a v e r a g e m o l e c u la r w eig h t and s e d i m e n t a t i o n c o e f f i c i e n t o f human lymphocyte DNA a f t e r d i f f e r e n t ti m e s o f c e l l s t o r a g e a r e g i v e n i n T ab . 1. The a v e r a g e m o l e c u l a r w eig h t o f DNA r e l e a s e d from lym p h ocy tes i a e q u a l to 2 6 .1 0 + 2 .8 7 x 10^ d a l t o n s .
T a b l e 1
E f f e c t o f s t o r a g e time o f ly m p h o cy te s upon th e a v e r a g e m o l e c u l a r w ei g h t and s e d i m e n t a ti o n c o e f f i c i e n t o f DNA
Wpływ c z a s u przechowywania l im f o cy tów na masę c z ą s te c z k o w ą i s t a ł ą s e d y m e n t a c j i DNA
BjiHHHHe xpaHeHHH jihmJoijhtob Ha MOJieKyjiapHuft Bec
u
KOHCTaHTy ceĄHMeHraiiHH HHK S t o r a g e tim e n X o 1 S e d i m e n t a ti o n c o e f f i c i e n t , S m + s l+ CÛ 0 h 11 2 6 . 1 0 + 2 .8 7 1 2 1 .0 + 4 . 9 24 h 10 5 . 0 8 + 0 .4 5 6 3 .1 + 2 . 5 48 h 9 2 .7 1 + 0 .4 7 4 8 .6 + 2 .6 T h is v a l u e c o r r e s p o n d s to e s t a b l i s h e d v a l u e s o f s u b u n it m o le c u la r w eig h t o f mammalian DNA i n a l k a l i n e s u c r o s e d e n s i t y g r a d i e n t s [ 5 , 2 1 , 2 6, 2 7 ] . The a v e r a g e m o l e c u l a r w e ig h t o f DNA r e l e a s e d from lym p h ocy tes s t o r e d f o r 24 h i s e q u a l to 5 . 0 8 + 0 .4 5 x lo"'* d a l t o n s , and o f DNA from lym p h ocy tes s t o r e d f o r 48 h to 2 .7 1 + 0 .4 7 x 10? d a l t o n s .As i t r e s u l t s from th e d a t a g iv e n i n T ab . 1, th e s t o r a g e o f ly m p ho cy tes r e s u l t s i n a d e c r e a s e o f m o l e c u l a r w eig h t o f c e l l DNA. The d e c r e a s e i n th e a v e r a g e m o l e c u la r w e ig h t o f lymphocyte
P i g . 1. S e d i m e n ta t i o n p r o f i l e s o f 3H - l a b e l l e d human lymphocy te DNA i n a 5 -2 0 $ a l k a l i n e s u c r o s e d e n s i t y g r a d i e n t a f t e r v a r i o u s ti m e s o f s t o r a g e o f l y m p h o c y t es . DNA was o b t a i n e d by l y s i n g o f ly m p h oc yte s f o r 30 min b e f o r e c e n t r i f u g a t i o n . L y s i n g s o l u t i o n s 0 . 5 M NaOH, 0 .1 M EDTA. C e n t r i f u g a t i o n : 35 00 0 rpm, 120 min, a b o u t 1 0 °C , SW-60 r o t o r . Lymphoc ytes s t o r e d f o r ---- O h , --- 24 h,
---48 h
P r o f i l e s e d y m e n t a c j i znakowanego tr y t em DNA li m f oc y tó w l u d z k i c h w 5-20% g r a d i e n c i e g ę s t o ś o i a l k a l i c z n e j s a c h a r o z y . DNA o trz ym a-no w wyniku 30-minutow ej l i z y l im fo c y tó w na p ow ier z c h n i g r a -d i e n t u b e z p o ś r e -d n i o p r z e-d wirowaniem. S k ł a d r o z t w o r u l i z u j ą c e g o : 0 , 5 M NaOH, 0 ,1 M EDTA. W irowanie: 35 000 rpm, 120 min, temp. 1 0 °C , r o t o r SW-60. L im f oc yty przechowywano p r z ez ----0 h , ---24 h,
--- 48 h
npo$HJIH CyflHMeHTailHH Me^ieHoił TpHTOM ,HHK qeJIOBe^ieCKHX JIHMjOIJHTOB B 5-20% rpa^HenTe iuiothocth nejiotiHoił ca xap o3 u. ,HHK nojiyuanH rtocjie 3 0 - M H H y r H o r o jiH3Hca ^hm$ol(htob Ha iiOBepxHOCTH rpa^HeHTa Henocpe^- cTBeHHo nepe^ i;eHTpn<i>yrnpoBaHKeM. CocTaB jiH3Hpy»mero pacTBopa: 0 ,5 M NaOH, 0 ,1 M EDTA. LleHTpmiiyrHpoBaHHe: 35 000 o6opotob/mhh, 120 M H H y i , TeMoepaTypa 1 0°C , poTop SW-60. JIhm$ouhtu xpaHHJiH b
DNA amounted to 81% f o r th e tim e i n t e r v a l o f 0 -2 4 h , to 90% f o r the time i n t e r v a l o f 0 -4 8 h , and to 47% f o r th e time i n t e r v a l o f 2 4 -4 8 h ( T a b . 2 ) . These c h a n g e s a r e s t a t i s t i c a l l y s i g n i f i c a n t .
T a b l e 2
D i f f e r e n c e s between th e a v e r a g e m o l e c u la r w e i g h t s o f DNA o b t a i n e d from ly m ph ooytes s t o r e d f o r d i f f e r e n t tim e i n t e r v a l s
Zmiany masy o z ą s te c z k o w e j DNA otrzy ma neg o z li m fo c y tó w przechowywanych p r z e z róż ny o k r e s c z a s u
H3M6HeHHH MOJieKyjiHpHoro Bec a HHK, nojiy'ieHHOft H3 jiHMi|>0iiHT0B xpaHHMhix B paajiH^iHhie npoMentyTKH BpeMeHH
Time i n t e r v a l s DNA m o l e c u la r w eig h t
c h a n g es ( i n per c e n t ) P<
0 h ----*» 24 h 81 0.0 0 1
0 h ----»• 48 h 90 0 .0 0 1
24 h ---- 48 h 47 0 .0 0 2
The lym phocyte s u r v i v a l , a s e s t i m a t e d on th e b a s i s o f Trypan B lu e s t a i n i n g amounted to 99% a f t e r a 24 h c u l t i v a t i o n . A f t e r 24 and 48 h o f s t o r a g e o f l y m p h o c y t e s , t h e i r v i a b i l i t y d e c r e a s e d to
90%.
A f t e r the same time o f s t o r a g e , th e amount o f lym p h oc ytes d e c r e a s e d by 5 ^ . I t seems t h a t a f t e r s t o r a g e o f ly m p h o c yt es in PBS ( p h o s p h a te b u f f e r e d s a l i n e ) a t 4 ° C , v i a b i l i t y and d e n s i t y o f th e c e l l s were c o n s e r v e d b e c a u s e th e o b se r v ed c h a n g e s a r e d e v oi d o f s t a t i s t i c a l s i g n i f i c a n c e . S i m i l a r r e s u l t s were o b t a i n e d i n ex -p e ri m en ts on c e l l s u r v i v a l e s t i m a t e d by m easurem ents o f ^ 1Cr r e l e a s e . D i s c u s s i o n % - T r i t i u m i n c o r p o r a t e d i n t o b i o l o g i c a l m o l e c u l e s can c a u s e damage e i t h e r due to b e t a - r a d i a t i o n o f t r i t i u m d e c a y , or from c h em ic a l c h a n g e s a s s o c i a t e d w it h the t r a n s m u t a t i o n from 3H to 3He. I t h a s been shown t h a t % - d e c a y s i n th e n u c l e u s a r e more e f f e c t i v e th an % - d e c a y s i n o y to p l a s m [ 5 ] . H o f e r et a l . [ 1 2 ] showed t h a t c y t o t o x i c e f f e c t s o f 6^ G a - c i t r a t e , th ym idi ne and ^ ^ I - i o d o d e o x y u r i d i n e on L 1210 le u ka e m ia c e l l s l a b e l l e d w ith v a r i o u s d o s e s o f t h e s e t h r e e r a d i o i s o t o p e s r e s u l t e x c l u s i v e l y from n u c l e a r damage. T h e r e f o r e -% -thymid ine d eca y demaged, f i r s to f a l l , DNA. T r l t i a t e d th y m id i n e ( -^HTdR) i s i n c o r p o r a t e d d i r e c t l y i n t o DNA and thua c o n se r v e d i n th e c e l l n u c l e u s a s a p a r t o f g e n e t i c m a t e r i a l [ 3 1 . For t h i s r e a s o n , i t seems t h a t i t i s ne-c e s s a r y to a v o i d s t o r a g e o f % - t h y m i d i n e - l a b e l l e d c e l l o u l t u r e a i n i n v e a t i g a t i o n a o f t h e i r DMA, b e c a u se s t o r a g e r e s u l t s i n a d e c r e a s e o f m o l e c u la r w eig h t o f t h i s DNA due to i t s d e g r a d a -t i o n . The r e a u l -t s o b t a i n e d i n t h i s paper i n d i c a t e that a v e r a g e m o le c u l a r w e ig h t o f DNA m arked ly d e c r e a s e s a f t e r 48 h s t o r a g e o f % - t h y m id i n e l a b e l l e d human ly m p h o c y t es . S i m i l a r l y , i n t r a c e l l u l a r 1^ C -d ec a ys i n l a b e l l e d V 79 C h in e se h am s te r c e l l s l e a d to s t r a n d b r e a k s and to a p r o g r e s s i v e d e c r e a s e i n m o l e c u la r w ei g h t o f DNA w ith i n c r e a s e d time o f s t o r a g e a t -1 9 6 °C [ 8 ] and 12'^I-d ec ay i n d u c e s d o u b l e - s t r a n d b r e a k s i n E. c o l l DNA when r a d i o l a b e l l e d b a c t e r i a were s t o r e d a t - 1 9 6 °C [ 1 8 ] , The s e d i m e n ta t i o n p r o f i l e s o f DNA o b t a in e d from HeLa c e l l s t r e a t e d w i th % - t h y m i d i n e waa a f f e c t e d by th e time o f e x p o s u r e o f t h e s e c e l l s to the r a d i o i s o -to p e [ 1 1 ] . However i t i s worth t o n o te t h a t some r e p o r t s s u -g -g e s t e d p o s s i b i l i t y o f su ch d e g r a d a t i o n c a u s e d by n o n r a d i o a c t i v e p r o c e s s e s [ 2 5 , 30 , 3 2 ] . In th e l i g h t o f t h e s e o b s e r v a t i o n the i n f l u e n c e o f enzymic p r o c e s s e s ca n not be e x c l u d e d . REFERENCES [ 1 ] B o y d E. , F e r c u s o n- S m i t h M. A. , 1/1 c D io u-g a l J . R. , G r i e g VV. R . , R a d i a t . R es . 57 , 482 ( 1 9 7 4 ) . [ 2 ] B r a d S . J . , l e y R a d i a t E. W., C h a n P. C. , A d e l . R e s . 6 4 , 555 ( 1 9 7 5 ). s t e i n [ 3 ] B u r k v e r i H. J . E. , J . , B u n k e r S . , R i t t e r M. R a d i a t . R e s . 6 2, 299 ( 1 9 7 5 ) . , c 1 e a-[ 4 ] B u r k i H. J . , M o u s t a c h i E . , R a d i a t . R e s . 7 1 , 635 ( 1 9 7 7 ) . [ 5 ] B u r k ( 1 9 6 8 ) . i H. J . , O k a d a S . , B i o p h y s . J . 8 , 445 [ 6 ] B u r k L. E . , i H. I n t . J J . , R o o t s R. , F e i n e n . R a d i a t . B i o l . 2 4 , 363 ( 1 9 7 3 ) . d e g e n
[7] B u r k i H. J . , ft 0 o t a R . , F e i n e n d e g e n L. E. , B o n d V. P ., I n t . J . R a d i a t . B i o l . 2 7 , 363 ( 1 9 7 5 ) . [ 8 ] C l e a v e r J . E . , B u r k i H. J . , I n t . J . R a d i a t . B i o l . 2 6 , 399 ( 1 9 7 4 ) . [9] C l e a v e r J . E. , T h o m a s G. H ., B u r k i H . J . S c i e n c e 177, 996 ( 1 9 7 3 ) . [1 0 ] D o n 1 0 n T . , N o r m a n A . , M u ta t. R e s . 1_3, 93 ( 1 9 7 1 ) . [ 1 1 ] H a b e n e r J . H. , B y n u m B. S . , S h a c k J . , B io c h im . B i o p h y s . A c ta 2 0 4 , 128 ( 1 9 6 9 ) . [ 1 2 ] H o f e r K. G. , H a r r i s C. R. , S m i t h J . M., I n t . J . R a d i a t . B i o l . 2 8 , 225 ( 1 9 7 5 ) . [1 3 ] J a g u s z t j nK r y n i c k a K., K u n i c k i G o l d -f i n g e r V/. J . H ., A c ta M i c r o b i o l . Po lon . 6_, 11 ( 1 9 7 4 ) . [ 1 4 ] J a g u s z t y n- K r y n i c k a K., K u n i c k i G o l d -f i n g e r W. J . H ., A c ta M i c r o b i o l . Po lon . io, 1 1 ( 1 9 7 4 ) . [ 1 5 ] K r i s c h R. E . , I n t . J . R a d i a t . B i o l . 2_1, 161 ( 1 9 7 2 ) . [1 6] K r i s c h R. E . , I n t . J . R a d i a t . B i o l . 2 5 , 261 ( 1 9 7 4 ) . [ 1 7 ] K r i s c h R. E . , I n t . J . R a d i a t . B i o l . 2 9 , 249 ( 1 9 7 6 ) . [ 1 8 ] K r i s c h R. E. , K r a s i n P . , S a u r i C. J . , I n t . J . R a d i a t . B i o l . 2 9 , 37 ( 1 9 7 6 ) . [ 1 9 ] K r i s c h R. E . , L e y R . D ., I n t . J . R a d i a t . B i o l . 2 5 , 21 ( 1 9 7 4 ) . [ 2 0 ] K r i s c h R. E . , S a u r i C. J . , I n t . J . R a d i a t . B i o l . 2 7 , 553 ( 1 9 7 5 ) . [ 2 1 ] L e t t J . T . , K l u c i s E . S . , S u n C . , B i o p h y s . J . 1 0, 277 ( 1 9 7 0 ) . [ 2 2 ] L e y R. D ., K r i s c h R. E . , I n t . J . R a d i a t . B i o l . 2 5 , 531 ( 1 9 7 4 ) . [ 2 3 ] L i a c 0 H . , L i s c o E . , A d e l s t e i n S. J . , B a n k s H. H . , I n t . J . R a d i a t . B i o l . 2 4 , 45 ( 1 9 7 3 ) . [ 2 4 ] I c G r a t h R. A . , W i l l i a m s R . W., N a t u re 2 1 2 , 534 ( 1 9 6 6 ) . [ 2 5 ] 0 c k e y C. H ., I n t . J . R a d i a t . B i o l . 13, 479 ( 1 9 6 7 ) . [2 6 ] 0 r m e r o d M. G ., L e h m a n A. R . , B io c h im . B i o p h y s . A c ta 2 4 7 , 369 ( 1 9 7 1 ) . [ 2 7 ] O r m e r o d M. G. , S t e v e n s U ., B i oc h im . B i o p h y s . A c ta 2 3 2 , 72 ( 1 9 7 1 ) .
10 B . R ózga , E . P o zi om sk a , A. Kooeva- Ch yla [ 2 8 ] P e t e r s o n S . , B o c k r a t h R. C . , J . Mol. B i o l . 13, 600 ( 1 9 6 5 ) . [2 9 ] S o h m i d t A . , H o t z G . , I n t . J . R a d i a t . B i o l . 2 4 , 307 ( 1 9 7 3 ) . [3 0 ] S h i p 1 e y W. U. , B a k e r A. R. , C o 1 t e n H, R. ,
J .
Immun. 106 , 576 ( 1 9 7 1 ) . [3 1 ]s t
u d i e r F . V/.,, J . Mol. B i o l . 1 1 , 373 ( 1 9 6 5 ) . [3 2 ] W i 1 1 i a m s J . R . , L i t t l eJ .
B . , R a d i a t . R e s . 55» 527 ( 1 9 7 3 ) . [3 3 ] W i 1 1 i a m s J . R . , L i t t l e J . B. , S h i p l e y W. U . , N a tur e 2 5 2 , 754 ( 1 9 7 4 ) . Department o f B i o p h y s i c s I n s t i t u t e o f B i o c h e m i st r y and B i o p h y s i o s U n i v e r s i t y o f ŁódźB ł a ż e j R ó zg a , E l ż b i e t a Poz ioms ka , A neta K oc e va -C h y ła
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DNA li m fo c y tó w l u d z k i c h z krwi obwodowej znakowano H -ty mid y- n ą . Oznaczano wpływ ^H-tymidyny na p rz e ż y w a ln o ść komórek i na w ł a ś c i w o ś c i se d y m en ta c y jn e DNA l im f oc y tó w przechowywanych w PBS w temp. 4°C p r z ez różn y o k r e s c z a s u . Masg c z ą st e c z k o w ą DNA^wyznacz ano metod ą u l t r a w i r o w a n i a w a l k a l i c z n y m g r a d i e n c i e g ę s t o s c i s a -c h a r o z y . S tw ie r d z o n o , ż e prze-chowywanie znakowanych 3H-tymidy ną li m fo c y tó w powoduje d e g r a d a c j ę i c h DNA. N ie zaobserwowano zmian w p r z e ż y w a l n o ś c i l i m f o c y tó w , co s u g e r u j e , że w s to sow a ny ch
warun-ka ch przechowywania u s z k o d z e n ie DNA p o p r z e d z a ś m ie r ć kom orki.
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BO BPEMH 'Ä X XPAHEHHH 3 HHK ' J e j i o B e n e c K H X j i h mJi o h h t o b n e p H t J i e p H i e c K o B k p o b h M e T M H H - t h - m h ą h h o m. O n p e ę e Ji H J iH B Ji n a H H e 3 h —t h m h J iH H a H a x p a H e H H e k j i ö t o k h H a c e n H M e H T a u H O H H b ie C B o M c T B a ĄHK jiM M fo u s iT O B , x p a H H M b ix b P B S ( Ó y $ e p H Ł iii p a c T B o p $ K 3 H O J i o r n M e c K o 8 c o j i ą ) n p a l e M t i e p a T y p e 4 C b p a a J iH U H u e n p o - M escy TK H B p e M e H H . M o Jie ic y jiH p H b ift B e e £ H K o n p e ^ e j i f l J i H m c t o ^ o m a n < M > e - p e H u n p o B a H H o r o y jife T p a u e H T p n iiiy r H p o B a H H H b m e jio u H O M r p a ,n H e H T e i u i o t -
h o c t h c a x a p o 3 H . r i0 K a 3 a H 0 , q TO x p a H e n n e j i h wJj o u h t o b, M e ^ e H u x } H - t h -
m h h h h o m B U 3 M B a e T p a c n a n HHK j i h m$o i;h t o b. C y m e c T B e H H h i e o t j i h m h h b n e - p e *H B a H K H K Jie T O K H e H a d j n o ^ a ^ H c b , m o c B H ą e T e J i b C T B y e T o t o m , u t o b n a H H H x y c J io B H H x x p a H e H o a n o B p e * f l e H H e ĄHK n p e u m e c T B y e T c M e p T H K J i e - T K H .
