15. 12. 1977
1651
Specialia
Effect of d i b u t y r y l c A M P and t h e o p h y l l i n e on c u l t u r e d rat e m b r y o n i c s h i e l d s N. S k r e b a n d Lj. H o f m a n
Department o[ Biology, Faculty o/Medicine, Salata 3, YU-41000 Zagreb (Yugoslavia), 7 October 1976 Summary. E f f e c t s of N 6, O 2 d i b u t y r y l a d e n o s i n e 3',5'-cyclic m o n o p h o s p h a t e (rib-cAMP) a n d t h e o p h y l l i n e (Th) on c u l t u r e d r a t e m b r y o n i c shields were s t u d i e d . A f t e r a d d i t i o n of d b - c A M P t o t h e c u l t u r e m e d i u m , a n increase of t h e w e i g h t of e x p l a n t s a n d of t h e incidence of t h e skeletal muscle was o b s e r v e d . T h e o p h y l l i n e s e e m s to be ineffective. Cyclic n u c l e o t i d e s are k n o w n t o c o n t r o l tile g r o w t h of m a m m a l i a n cells in cell c u l t u r e 1 a n d t h e d i f f e r e n t i a t i o n of d i f f e r e n t e m b r y o n i c cells~, d b - c A M P can s t i m u l a t e t h e cell g r o w t h a n d a c c e l e r a t e t h e a p p e a r e n c e of g a m m a c r y s t a l l i n s in t h e m o n o l a y e r c u l t u r e of r a t lens epithelial cells ~. I n m o u s e n e u r o b l a s t o m a cells in vitro, t h e s a m e cyclic n u c l e o t i d e i r r e v e r s i b l y i n d u c e s several d i f f e r e n t i a t e d f u n c t i o n s c h a r a c t e r i s t i c of m a t u r e n e u r o n s 4. F i n a l l y a n e u r a l i z i n g influence of d b - c A M P o n t h e u n d e t e r m i n e d a m p h i b i a n 5 or c h i c k 6 e c t o d e r m h a s r e c e n t l y b e e n o b s e r v e d . Tile m o d i f i e d o r g a n c u l t u r e of r a t e m b r y o n i c shields was s h o w n to p r o v i d e f a v o u r a b l e c o n d i t i o n s n e c e s s a r y for t h e d i f f e r e n t i a t i o n of m a i n tissues 7. H o w e v e r , t h e histological d i f f e r e n t i a t i o n in e x p l a n t s p r o v e d inferior to t h a t obt a i n e d in h o m o g r a f t s of t h e s a m e shields u n d e r t h e k i d n e y capsule. T h e p u r p o s e of t h e p r e s e n t e x p e r i m e n t was t o f i n d o u t w h e t h e r t h e a d d i t i o n of d b - c A M P a n d / o r T h to the culture medium can improve the phenotypic e x p r e s s i o n of whole r a t e m b r y o n i c shields as d e s c r i b e d a b o v e for d i f f e r e n t v e r t e b r a t e cells in vitro. Materials and methods. F e m a l e r a t s of t h e i n b r e d F i s c h e r s t r a i n were killed a f t e r 9 d a y s of p r e g n a n c y a n d t h e e g g - c y l i n d e r s a t tile p r i m i t i v e s t r e a k s t a g e were isolated. T h e e x t r a e m b r y o n i c p a r t was c u t off a t tile level of t h e a m n i o n a n d t h e shields were p u t o n t h e lens p a p e r supp o r t e d b y a stainless steel grid placed in an e m b r y o l o g i c a l w a t c h glass as d e s c r i b e d p r e v i o u s l y L T h e liquid m e d i u m c o n s i s t e d of E a g l e ' s m i n i m u m essential m e d i u m supp l e m e n t e d w i t h 40% of r a t serum. F r o m 5 to 14 d a y s db-cAiVfP (Sigma), T h or b o t h a g e n t s t o g e t h e r were a d d e d t o t h e m e d i u m in t h e c o n c e n t r a t i o n of 10 -8 M. A f t e r 14 d a y s t h e e x p l a n t s were fixed, a n d histological sections were e x a m i n e d .
mControl
~Th
. . . . . . . . . . . . o/
-cAMP+Th
Results and discussion. F r o m t h e figure one c a n see t h a t t h e g u t e p i t h e l i u m a p p e a r s m o r e f r e q u e n t l y in t h e t r e a t e d series t h a n in c o n t r o l s (Z2 = 7.4; p < 0.01). H o w e v e r , before t h e e v a l u a t i o n of t h i s result, p r e v i o u s d a t a h a v e t o b e t a k e n into c o n s i d e r a t i o n . I n all u n t r e a t e d series, o b t a i n e d so far (from 350 e x p l a n t s ) t h e p e r c e n t a g e of t h e g u t e p i t h e l i u m was t h e s a m e as in t h e series t r e a t e d w i t h d b - c A M P (60%). This r e s u l t m u s t t h e r e f o r e be i n t e r p r e t e d w i t h g r e a t caution, in s p i t e of t h e f a c t t h a t we c u l t i v a t e d parallel series w i t h a n d w i t h o u t rib-cAMP. O n t h e o t h e r h a n d t h e skeletal muscle, w h i c h also a p p e a r s m o r e f r e q u e n t l y in t h e t r e a t e d series t h a n in controls (Z 2 = 40.65; p < 0.001), seems t o r e f l e c t t h e real situation. Our p r e v i o u s e x p e r i m e n t s are in full a g r e e m e n t w i t h r e c e n t c o n t r o l series. T h e skeletal muscle is always rare, its incidence n e v e r e x c e e d i n g 20% (in 400 explants). B u t y r i c acid e i t h e r h a d a t o x i c effect (10 -2 M) or h a d no effect w h a t s o e v e r (10 -a M). F o r t h e t i m e b e i n g it is difficult to give a n y plausible e x p l a n a t i o n for t h i s action of d b - c A M P on t h e a p p e a r a n c e of t h e skeletal muscle. T h e o p h y l l i n e , w h i c h is k n o w n to increase tile intracellular pool of cyclic A M P t h r o u g h p h o s p h o d i e s t e r a s e inhibition, u s u a l l y acts in tile s a m e w a y as t h e a d d i t i o n of d b - c A M P , while in our s y s t e m T h seems t o h a v e b e e n ineffective. H o w e v e r , results similar to ours h a v e b e e n o b t a i n e d in d i f f e r e n t s y s t e m s w h e n T h h a d no visible effect 3,6. Furthermore, db-cAMP and Th proved to inhibit the cell g r o w t h a n d t h e fusion of m o n o n u e l e a t e d m y o b l a s t s i n t o m u l t i n u c l e a t e d m y o t u b e s in a m o n o l a y e r culture s. I n our e x p e r i m e n t , we o b t a i n e d j u s t t h e o p p o s i t e effect. T h e p e r c e n t a g e of m y o t u b e s in e x p l a n t s a f t e r a d d i t i o n of d b - c A M P was h i g h e r t h a n in controls. Similar results were r e c e n t l y p u b l i s h e d a n d it was claimed t h a t , u n d e r t h e p r o p e r conditions, e x p e r i m e n t a l elevation in c A M P can in f a c t s t i m u l a t e m y o b l a s t fusion 9. D u r i n g our e x p e r i m e n t s , we o b s e r v e d t h a t t h e surface of t h e histological sections of t r e a t e d series w a s larger t h a n t h a t in controls. T h e r e f o r e we w e i g h e d s o m e e x p l a n t s before fixation. The w e i g h t of t h e c o n t r o l series was 0.217 • 0.25 g (n = 29), a n d in t h e p r e s e n c e of d b - c A M P : 0.401 -4- 0.022 g (n = 27). The difference is s t a t i s t i c a l l y h i g h l y s i g n i f i c a n t (t = 5.79; p < 0.001). I n our p r e v i o u s e x p e r i m e n t s we h a d o b s e r v e d t h a t , d u r i n g t h e first week of culture, t h e g r o w t h of e x p l a n t s was clearly visible,
1 2 3 4 5 ,o Epidermis
Brain
Gut
Skeletalmuscle Cartilage Glands
Incidence of tissues found in explants of rat embryonic shields a f t e r 2 weeks of culture. Control series = 112 explants; 10 -8 M db-cAMP = 62 explants; 10-3 M theophylline= 63 explants; db-cAMP and Th = 77 explants.
6 7 8 9
R.W. Holley, Nature, Lond. 258, 487 (1975). D. McMahon, Science 185, 1012 (1974). M.O. Creighton and J. R. Trevithick, Nature, Lond. 2d9, 767 (1974). K.N. Prasad, Differentiation 2, 367 (1974). H . L . Wahn, L. E. Lightbody, T. T. Tchen and J. D. Taylor, Science 188, 366 (1975). B. Bjerke, Experientia 30, 534 (1974). N. ~kreb and A. Svajger, Wilhelm Roux' Arch. 173, 228 (1973). J . P . Wahrmann, R. Winand and D. Luzzati, Nature New Biol. 245, 112 (1973). D.L. Friedman, Physiol. Rev. 56, 652 (1976).
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while in t h e s e c o n d w e e k t h e e x p l a n t s g r a d u a l l y b e c a m e smaller. W h e n m e a s u r i n g t h e a m o u n t of D N A , R N A a n d p r o t e i n , t h e c u r v e i n d i c a t e d t h e s a m e decline d u r i n g t h e second w e e k ( u n p u b l i s h e d results). T h e a c t i o n of d b c A M P m i g h t t h e r e f o r e b e e x p l a i n e d b y t h e i n h i b i t i o n of cell d e a t h / o r t h e s t i m u l a t i o n of cell p r o l i f e r a t i o n . W e
EXPERIENTIA 33[12
m u s t b e a r in m i n d t h a t t h e g r o w t h - p r o m o t i n g effects of c A M P are s o m e t i m e s c l a i m e d t o b e d u e t o t h e r e s t o r a t i o n of t h e p u r i n e n u c l e o t i d e pool a n d n o t to a specific influence 1% 10 T. Hovi, A. Vaheri, Nature New Biol. 245, 175 (1973).
D i f f e r e n c e s in c y t o c h a l a s i n D - i n d u c e d s u r f a c e a l t e r a t i o n s b e t w e e n c h r o n i c lymphocytic leukaemic and normal lymphocytes 1 L. S k i n n i d e r
Department o/Pathology, University o/Saskatchewan, Saskatoon S7N OWO (Canada), 9 May 1977 Summary. C y t o c h a l a s i n D (CD) causes a n u n u s u a l surface a l t e r a t i o n in n o r m a l l y m p h o c y t e s c o n s i s t i n g of t h e f o r m a t i o n of focal i r r e g u l a r c l u b - s h a p e d cell processes. L y m p h o c y t e s f r o m c h r o n i c l y m p h o c y t i c l e u k a e m i c cases d i d n o t s h o w t h i s c h a n g e o n e x p o s u r e t o CD. T h e r e was e i t h e r n o surface c h a n g e or, i n some cases, clear d o u b l e - m e m b r a n e lined vesicles were f o r m e d a n d a p p e a r e d to b e d i s c h a r g e d f r o m t h e cell. T h i s difference in r e s p o n s e m a y b e r e l a t e d t o t h e c h a n g e s in cell m e m b r a n e s k n o w n t o o c c u r in m a l i g n a n t t r a n s f o r m a t i o n . T h e effect of c y t o c h a l a s i n s o n m a m m a l i a n cells are n u m e r o u s a n d are t h o u g h t t o b e b r o u g h t a b o u t b y a n a l t e r a t i o n of t h e cell m i c r o f i l a m e n t f u n c t i o n e-5. S t u d i e s w i t h t r i t i a t e d c y t o c h a l a s i n D (CD) s u p p o r t t h i s c o n c e p t as it is t a k e n u p a n d b o u n d t o t h e s u b p l a s m a l e m m a l m i c r o f i l a m e n t s or cell m e m b r a n e % P r e v i o u s u l t r a s t r u c r u r a l s t u d y o n t h e effects of CD o n t h e m o r p h o l o g y of c u l t u r e d t u m o u r cells s h o w e d c y t o p l a s m i c p r o j e c t i o n s t h o u g h t t o b e s i m i l a r t o zeiotic b l e b s L T h e p r e s e n t s t u d y of t h e effects of CD o n n o r m a l h u m a n p e r i p h e r a l b l o o d l y m p h o c y t e s s h o w t h e m o r p h o l o g i c a l surface a l t e r a t i o n s t o c o n s i s t of u n u s u a l c y t o p l a s m i c processes. T h e s e processes w e r e focal a n d did n o t u n d e r g o r e p e a t e d p r o t r u s i o n a n d r e t r a c t i o n in c o n t r a s t t o t h e p h e n o m e n o n of zeiosis s. T h e c h a n g e seen was in k e e p i n g w i t h t h e c o n c e p t of t h e a c t i o n of t h e c y t o c h a l a s i n s o n t h e m i c r o f i l a m e n t s . I n t h e cases of c h r o n i c l y m p h o c y t i c l e u k a e m i a (CLL) t h e r e w a s e i t h e r n o m a r k e d surface c h a n g e or, in a few cases, t h e r e was f o r m a t i o n of d o u b l e m e m b r a n e lined vesicles a t t h e cell surface. T h e r e a s o n for t h e d i f f e r e n t m o r p h o l o g i c a l r e s p o n s e in t h e c h r o n i c l y m p h o c y t i c l e u k a e m i c l y m p h o cytes was not apparent from this study.
Fig. 1. Arrows point to focal cytoplasmic irregularities at 1 pole of normal lymphocytes exposed to CD 15 Exg/ml, unfixed wet specimen. • 550.
Materials and methods. CD was dissolved in d i m e t h y l sulfoxide (DMSO). L y m p h o c y t e s were o b t a i n e d f r o m n o r m a l d o n o r s a n d f r o m cases of CLL. T h e following l y m p h o c y t e p r e p a r a t i o n s were used. 1. L y m p h o c y t e s in l e u c o c y t e - r i c h p l a s m a w i t h o u t f u r t h e r m a n i p u l a t i o n , 2. lymphocytes separated on a Ficoll-Hypaque gradient, w a s h e d a n d s u s p e n d e d in g r o w t h m e d i u m (GM) cons i s t i n g of E a g l e ' s m e d i u m w i t h 10~o f e t a l calf serum, 3. l y m p h o c y t e s s e p a r a t e d on a F i c o l l - H y p a q u e g r a d i e n t , w a s h e d w i t h GM a n d r e s u s p e n d e d in p l a s m a . Cell susp e n s i o n s a t a c o n c e n t r a t i o n of 1 million cells p e r m l were i n c u b a t e d for 40 m i n a t 37 ~ w i t h a f i n a l c o n c e n t r a t i o n of 1. CD, 1 ~xg/ml a n d 2. CD, 15 vg/ml. T h e c o n c e n t r a t i o n of D M S O was 0.5%. C o n t r o l s w i t h a n d w i t h o u t 0.5% D M S O were also p r e p a r e d . U n f i x e d w e t p r e p a r a t i o n s were s t u d i e d i m m e d i a t e l y a t t h e e n d of t h e 40 m i n i n c u b a t i o n b y p h a s e m i c r o s c o p y . T h e cell s u s p e n s i o n s were t h e n fixed in ice-cold 2 % g l u t e r a l d e h y d e for s c a n n i n g e l e c t r o n
Fig. 2. TEM of above preparation showing surface irregularities to be cytoplasmic, club-shaped processes. • 21,000.
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Specialia
Effect of d i b u t y r y l c A M P and t h e o p h y l l i n e on c u l t u r e d rat e m b r y o n i c s h i e l d s N. S k r e b a n d Lj. H o f m a n
Department o[ Biology, Faculty o/Medicine, Salata 3, YU-41000 Zagreb (Yugoslavia), 7 October 1976 Summary. E f f e c t s of N 6, O 2 d i b u t y r y l a d e n o s i n e 3',5'-cyclic m o n o p h o s p h a t e (rib-cAMP) a n d t h e o p h y l l i n e (Th) on c u l t u r e d r a t e m b r y o n i c shields were s t u d i e d . A f t e r a d d i t i o n of d b - c A M P t o t h e c u l t u r e m e d i u m , a n increase of t h e w e i g h t of e x p l a n t s a n d of t h e incidence of t h e skeletal muscle was o b s e r v e d . T h e o p h y l l i n e s e e m s to be ineffective. Cyclic n u c l e o t i d e s are k n o w n t o c o n t r o l tile g r o w t h of m a m m a l i a n cells in cell c u l t u r e 1 a n d t h e d i f f e r e n t i a t i o n of d i f f e r e n t e m b r y o n i c cells~, d b - c A M P can s t i m u l a t e t h e cell g r o w t h a n d a c c e l e r a t e t h e a p p e a r e n c e of g a m m a c r y s t a l l i n s in t h e m o n o l a y e r c u l t u r e of r a t lens epithelial cells ~. I n m o u s e n e u r o b l a s t o m a cells in vitro, t h e s a m e cyclic n u c l e o t i d e i r r e v e r s i b l y i n d u c e s several d i f f e r e n t i a t e d f u n c t i o n s c h a r a c t e r i s t i c of m a t u r e n e u r o n s 4. F i n a l l y a n e u r a l i z i n g influence of d b - c A M P o n t h e u n d e t e r m i n e d a m p h i b i a n 5 or c h i c k 6 e c t o d e r m h a s r e c e n t l y b e e n o b s e r v e d . Tile m o d i f i e d o r g a n c u l t u r e of r a t e m b r y o n i c shields was s h o w n to p r o v i d e f a v o u r a b l e c o n d i t i o n s n e c e s s a r y for t h e d i f f e r e n t i a t i o n of m a i n tissues 7. H o w e v e r , t h e histological d i f f e r e n t i a t i o n in e x p l a n t s p r o v e d inferior to t h a t obt a i n e d in h o m o g r a f t s of t h e s a m e shields u n d e r t h e k i d n e y capsule. T h e p u r p o s e of t h e p r e s e n t e x p e r i m e n t was t o f i n d o u t w h e t h e r t h e a d d i t i o n of d b - c A M P a n d / o r T h to the culture medium can improve the phenotypic e x p r e s s i o n of whole r a t e m b r y o n i c shields as d e s c r i b e d a b o v e for d i f f e r e n t v e r t e b r a t e cells in vitro. Materials and methods. F e m a l e r a t s of t h e i n b r e d F i s c h e r s t r a i n were killed a f t e r 9 d a y s of p r e g n a n c y a n d t h e e g g - c y l i n d e r s a t tile p r i m i t i v e s t r e a k s t a g e were isolated. T h e e x t r a e m b r y o n i c p a r t was c u t off a t tile level of t h e a m n i o n a n d t h e shields were p u t o n t h e lens p a p e r supp o r t e d b y a stainless steel grid placed in an e m b r y o l o g i c a l w a t c h glass as d e s c r i b e d p r e v i o u s l y L T h e liquid m e d i u m c o n s i s t e d of E a g l e ' s m i n i m u m essential m e d i u m supp l e m e n t e d w i t h 40% of r a t serum. F r o m 5 to 14 d a y s db-cAiVfP (Sigma), T h or b o t h a g e n t s t o g e t h e r were a d d e d t o t h e m e d i u m in t h e c o n c e n t r a t i o n of 10 -8 M. A f t e r 14 d a y s t h e e x p l a n t s were fixed, a n d histological sections were e x a m i n e d .
mControl
~Th
. . . . . . . . . . . . o/
-cAMP+Th
Results and discussion. F r o m t h e figure one c a n see t h a t t h e g u t e p i t h e l i u m a p p e a r s m o r e f r e q u e n t l y in t h e t r e a t e d series t h a n in c o n t r o l s (Z2 = 7.4; p < 0.01). H o w e v e r , before t h e e v a l u a t i o n of t h i s result, p r e v i o u s d a t a h a v e t o b e t a k e n into c o n s i d e r a t i o n . I n all u n t r e a t e d series, o b t a i n e d so far (from 350 e x p l a n t s ) t h e p e r c e n t a g e of t h e g u t e p i t h e l i u m was t h e s a m e as in t h e series t r e a t e d w i t h d b - c A M P (60%). This r e s u l t m u s t t h e r e f o r e be i n t e r p r e t e d w i t h g r e a t caution, in s p i t e of t h e f a c t t h a t we c u l t i v a t e d parallel series w i t h a n d w i t h o u t rib-cAMP. O n t h e o t h e r h a n d t h e skeletal muscle, w h i c h also a p p e a r s m o r e f r e q u e n t l y in t h e t r e a t e d series t h a n in controls (Z 2 = 40.65; p < 0.001), seems t o r e f l e c t t h e real situation. Our p r e v i o u s e x p e r i m e n t s are in full a g r e e m e n t w i t h r e c e n t c o n t r o l series. T h e skeletal muscle is always rare, its incidence n e v e r e x c e e d i n g 20% (in 400 explants). B u t y r i c acid e i t h e r h a d a t o x i c effect (10 -2 M) or h a d no effect w h a t s o e v e r (10 -a M). F o r t h e t i m e b e i n g it is difficult to give a n y plausible e x p l a n a t i o n for t h i s action of d b - c A M P on t h e a p p e a r a n c e of t h e skeletal muscle. T h e o p h y l l i n e , w h i c h is k n o w n to increase tile intracellular pool of cyclic A M P t h r o u g h p h o s p h o d i e s t e r a s e inhibition, u s u a l l y acts in tile s a m e w a y as t h e a d d i t i o n of d b - c A M P , while in our s y s t e m T h seems t o h a v e b e e n ineffective. H o w e v e r , results similar to ours h a v e b e e n o b t a i n e d in d i f f e r e n t s y s t e m s w h e n T h h a d no visible effect 3,6. Furthermore, db-cAMP and Th proved to inhibit the cell g r o w t h a n d t h e fusion of m o n o n u e l e a t e d m y o b l a s t s i n t o m u l t i n u c l e a t e d m y o t u b e s in a m o n o l a y e r culture s. I n our e x p e r i m e n t , we o b t a i n e d j u s t t h e o p p o s i t e effect. T h e p e r c e n t a g e of m y o t u b e s in e x p l a n t s a f t e r a d d i t i o n of d b - c A M P was h i g h e r t h a n in controls. Similar results were r e c e n t l y p u b l i s h e d a n d it was claimed t h a t , u n d e r t h e p r o p e r conditions, e x p e r i m e n t a l elevation in c A M P can in f a c t s t i m u l a t e m y o b l a s t fusion 9. D u r i n g our e x p e r i m e n t s , we o b s e r v e d t h a t t h e surface of t h e histological sections of t r e a t e d series w a s larger t h a n t h a t in controls. T h e r e f o r e we w e i g h e d s o m e e x p l a n t s before fixation. The w e i g h t of t h e c o n t r o l series was 0.217 • 0.25 g (n = 29), a n d in t h e p r e s e n c e of d b - c A M P : 0.401 -4- 0.022 g (n = 27). The difference is s t a t i s t i c a l l y h i g h l y s i g n i f i c a n t (t = 5.79; p < 0.001). I n our p r e v i o u s e x p e r i m e n t s we h a d o b s e r v e d t h a t , d u r i n g t h e first week of culture, t h e g r o w t h of e x p l a n t s was clearly visible,
1 2 3 4 5 ,o Epidermis
Brain
Gut
Skeletalmuscle Cartilage Glands
Incidence of tissues found in explants of rat embryonic shields a f t e r 2 weeks of culture. Control series = 112 explants; 10 -8 M db-cAMP = 62 explants; 10-3 M theophylline= 63 explants; db-cAMP and Th = 77 explants.
6 7 8 9
R.W. Holley, Nature, Lond. 258, 487 (1975). D. McMahon, Science 185, 1012 (1974). M.O. Creighton and J. R. Trevithick, Nature, Lond. 2d9, 767 (1974). K.N. Prasad, Differentiation 2, 367 (1974). H . L . Wahn, L. E. Lightbody, T. T. Tchen and J. D. Taylor, Science 188, 366 (1975). B. Bjerke, Experientia 30, 534 (1974). N. ~kreb and A. Svajger, Wilhelm Roux' Arch. 173, 228 (1973). J . P . Wahrmann, R. Winand and D. Luzzati, Nature New Biol. 245, 112 (1973). D.L. Friedman, Physiol. Rev. 56, 652 (1976).
1652
Specialia
while in t h e s e c o n d w e e k t h e e x p l a n t s g r a d u a l l y b e c a m e smaller. W h e n m e a s u r i n g t h e a m o u n t of D N A , R N A a n d p r o t e i n , t h e c u r v e i n d i c a t e d t h e s a m e decline d u r i n g t h e second w e e k ( u n p u b l i s h e d results). T h e a c t i o n of d b c A M P m i g h t t h e r e f o r e b e e x p l a i n e d b y t h e i n h i b i t i o n of cell d e a t h / o r t h e s t i m u l a t i o n of cell p r o l i f e r a t i o n . W e
EXPERIENTIA 33[12
m u s t b e a r in m i n d t h a t t h e g r o w t h - p r o m o t i n g effects of c A M P are s o m e t i m e s c l a i m e d t o b e d u e t o t h e r e s t o r a t i o n of t h e p u r i n e n u c l e o t i d e pool a n d n o t to a specific influence 1% 10 T. Hovi, A. Vaheri, Nature New Biol. 245, 175 (1973).
D i f f e r e n c e s in c y t o c h a l a s i n D - i n d u c e d s u r f a c e a l t e r a t i o n s b e t w e e n c h r o n i c lymphocytic leukaemic and normal lymphocytes 1 L. S k i n n i d e r
Department o/Pathology, University o/Saskatchewan, Saskatoon S7N OWO (Canada), 9 May 1977 Summary. C y t o c h a l a s i n D (CD) causes a n u n u s u a l surface a l t e r a t i o n in n o r m a l l y m p h o c y t e s c o n s i s t i n g of t h e f o r m a t i o n of focal i r r e g u l a r c l u b - s h a p e d cell processes. L y m p h o c y t e s f r o m c h r o n i c l y m p h o c y t i c l e u k a e m i c cases d i d n o t s h o w t h i s c h a n g e o n e x p o s u r e t o CD. T h e r e was e i t h e r n o surface c h a n g e or, i n some cases, clear d o u b l e - m e m b r a n e lined vesicles were f o r m e d a n d a p p e a r e d to b e d i s c h a r g e d f r o m t h e cell. T h i s difference in r e s p o n s e m a y b e r e l a t e d t o t h e c h a n g e s in cell m e m b r a n e s k n o w n t o o c c u r in m a l i g n a n t t r a n s f o r m a t i o n . T h e effect of c y t o c h a l a s i n s o n m a m m a l i a n cells are n u m e r o u s a n d are t h o u g h t t o b e b r o u g h t a b o u t b y a n a l t e r a t i o n of t h e cell m i c r o f i l a m e n t f u n c t i o n e-5. S t u d i e s w i t h t r i t i a t e d c y t o c h a l a s i n D (CD) s u p p o r t t h i s c o n c e p t as it is t a k e n u p a n d b o u n d t o t h e s u b p l a s m a l e m m a l m i c r o f i l a m e n t s or cell m e m b r a n e % P r e v i o u s u l t r a s t r u c r u r a l s t u d y o n t h e effects of CD o n t h e m o r p h o l o g y of c u l t u r e d t u m o u r cells s h o w e d c y t o p l a s m i c p r o j e c t i o n s t h o u g h t t o b e s i m i l a r t o zeiotic b l e b s L T h e p r e s e n t s t u d y of t h e effects of CD o n n o r m a l h u m a n p e r i p h e r a l b l o o d l y m p h o c y t e s s h o w t h e m o r p h o l o g i c a l surface a l t e r a t i o n s t o c o n s i s t of u n u s u a l c y t o p l a s m i c processes. T h e s e processes w e r e focal a n d did n o t u n d e r g o r e p e a t e d p r o t r u s i o n a n d r e t r a c t i o n in c o n t r a s t t o t h e p h e n o m e n o n of zeiosis s. T h e c h a n g e seen was in k e e p i n g w i t h t h e c o n c e p t of t h e a c t i o n of t h e c y t o c h a l a s i n s o n t h e m i c r o f i l a m e n t s . I n t h e cases of c h r o n i c l y m p h o c y t i c l e u k a e m i a (CLL) t h e r e w a s e i t h e r n o m a r k e d surface c h a n g e or, in a few cases, t h e r e was f o r m a t i o n of d o u b l e m e m b r a n e lined vesicles a t t h e cell surface. T h e r e a s o n for t h e d i f f e r e n t m o r p h o l o g i c a l r e s p o n s e in t h e c h r o n i c l y m p h o c y t i c l e u k a e m i c l y m p h o cytes was not apparent from this study.
Fig. 1. Arrows point to focal cytoplasmic irregularities at 1 pole of normal lymphocytes exposed to CD 15 Exg/ml, unfixed wet specimen. • 550.
Materials and methods. CD was dissolved in d i m e t h y l sulfoxide (DMSO). L y m p h o c y t e s were o b t a i n e d f r o m n o r m a l d o n o r s a n d f r o m cases of CLL. T h e following l y m p h o c y t e p r e p a r a t i o n s were used. 1. L y m p h o c y t e s in l e u c o c y t e - r i c h p l a s m a w i t h o u t f u r t h e r m a n i p u l a t i o n , 2. lymphocytes separated on a Ficoll-Hypaque gradient, w a s h e d a n d s u s p e n d e d in g r o w t h m e d i u m (GM) cons i s t i n g of E a g l e ' s m e d i u m w i t h 10~o f e t a l calf serum, 3. l y m p h o c y t e s s e p a r a t e d on a F i c o l l - H y p a q u e g r a d i e n t , w a s h e d w i t h GM a n d r e s u s p e n d e d in p l a s m a . Cell susp e n s i o n s a t a c o n c e n t r a t i o n of 1 million cells p e r m l were i n c u b a t e d for 40 m i n a t 37 ~ w i t h a f i n a l c o n c e n t r a t i o n of 1. CD, 1 ~xg/ml a n d 2. CD, 15 vg/ml. T h e c o n c e n t r a t i o n of D M S O was 0.5%. C o n t r o l s w i t h a n d w i t h o u t 0.5% D M S O were also p r e p a r e d . U n f i x e d w e t p r e p a r a t i o n s were s t u d i e d i m m e d i a t e l y a t t h e e n d of t h e 40 m i n i n c u b a t i o n b y p h a s e m i c r o s c o p y . T h e cell s u s p e n s i o n s were t h e n fixed in ice-cold 2 % g l u t e r a l d e h y d e for s c a n n i n g e l e c t r o n
Fig. 2. TEM of above preparation showing surface irregularities to be cytoplasmic, club-shaped processes. • 21,000.
