15. 1. 1978

Specialia

were p r e i n c u b a t e d a t 37~ for 1 min. W G A (0.01 mg] 0.1 m l of isotonic NaC1-Tris) was a d d e d to a g g l u t i n a t e t h e ceils a n d a g g l u t i n a t i o n was o b s e r v e d w i t h i n 5 min. To s t u d y t h e effectiveness of a g g l u t i n a t i o n in vivo, L1~10 m o u s e l e u k e m i c ceils (1 • cells/mouse) were i n o c u l a t e d i.p. into B D F a mice. A f t e r 24 h t h e mice r e c e i v e d t h e 1st of 5 d a i l y i n j e c t i o n s of t h e d a u n o m y c i n e n t r a p p e d e r y t h r o c y t e s a n d W G A a t i n t e r v a l s of 10 min. Results and d~sc~ssion. 1 m l of resealed e r y t h r o c y t e s e n t r a p p e d a b o u t 4 m g of d a u n o m y c i n . R e s e a l e d e r y t h r o c y t e s (0.5 ml) were s u s p e n d e d in 5 m l of isotonic NaC1Tris a t 37 ~ a n d efflux of d a u n o m y c i n was e s t i m a t e d a t 1, 3, 6, 12 a n d 24 h a n d 23, 42, 78 a n d 8 4 % of e n t r a p p e d d a u n o m y c i n was l e a k e d o u t respectively. W G A a g g l u t i n a t e d t h e L1210 l e u k e m i c cells a n d resealed e r y t h r o c y t e s in v i t r o (figure 2). T h e g r e a t e s t increase in s u r v i v a l t i m e was o b t a i n e d in v i v o w h e n t h e d a u n o m y c i n e n t r a p p e d e r y t h r o c y t e s a n d W G A were g i v e n (table).

95

W G A m e d i a t e s a t t a c h m e n t of resealed e r y t h r o c y t e s t o t u m o r cells. T h e effectiveness of d a u n o m y c i n e n t r a p p e d e r y t h r o c y t e s a g a i n s t l e u k e m i c ceils especially d e m a n d s t a r g e t i n g of t h e e r y t h r o c y t e s to l e u k e m i c cells, a n d i t m a y b e possible t h r o u g h t h e use of t h e l e c t i n s w h i c h do n o t h a v e a g g l u t i n a b i l i t y of n o r m a l e r y t h r o c y t e s a n d mitogenic activity to lymphoyctes but have agglutinab i l i t y of l e u k e m i c cells a n d surface m o d i f i e d e r y t h r o c y t e s .

1 S.J. Updike, R. T. Wakiyama and E. N. Lightfoot, Jr, Science 193, 681 (1976). 2 J.C. Aub, C. Tieslau and A. Lankester, Proe. nat. Aead. Sci. USA 50, 613 (1963}. 3 H. Bodemann and H. Passow, J. Membr. Biol. 8, I (1972). 4 J. Bernard, R. Paul, M. Boiron, C. Jacquilliet and R. Marel, in: Rubidomycin, p. 12. Springer-Verlag, Berlin 1969. 5 E. Beutler, in: Red Cell Metabolism, 2nd ed, p. 11. G r u n e & Stratton, New York 1975.

E v i d e n c e f o r t h e p r e s e n c e of v i a b l e e n d o t h e l i a l c e l l s in c u l t u r e s d e r i v e d f r o m d i s s o c i a t e d r a t b r a i n 1 P. P a n u l a , F. J o 6 ~ a n d L. R e c h a r d t

Department o/Anatomy, University o/Helsinki, Siltavuocenpenger 20 b A, SF-O0170 Helsinki 17 (Finland), 8 August 1977 Summary. T h e m o r p h o l o g y a n d h i s t o c h e m i s t r y of d i s s o c i a t e d n e w b o r n r a t b r a i n was s t u d i e d in tissue culture. D i r e c t m i c r o s c o p y of d e v e l o p i n g ceils, electron m i c r o s c o p y a n d t h C a l k a l i n e p h o s p h a t a s e a c t i v i t y were used to i d e n t i f y t h e c a p i l l a r y e n d o t h e l i a l cells. Tissue c u l t u r e s o b t a i n e d from m e c h a n i c a l l y dissociated b r a i n tissue h a v e b e e n used for m a n y yearsa. As a cons e q u e n c e t h e r e is a c u m u l a t i v e i n f o r m a t i o n on t h e dist i n c t i v e p r o p e r t i e s of n e u r o n a l a n d glial cells, a n d on t h e e x t e n t a n d n a t u r e of g l i a l - n e u r o n a l i n t e r a c t i o n s . H o w e v e r , n o n e of t h e r e p o r t s p u b l i s h e d so far h a v e d e a l t seriously w i t h t h e p o s s i b i l i t y t h a t in dissociated cultures, t h e cells d e r i v i n g f r o m t h e b r a i n capillaries can also grow u n d e r t h e s a m e c o n d i t i o n s . I n t h e p r e s e n t paper, we s h o w h o w t h e c a p i l l a r y f r a g m e n t s o b t a i n e d b y m e c h a n i c a l d i s r u p t i o n of b r a i n tissue as inevitable contamination can undergo morphological c h a n g e s in vitro, a n d r e s u l t in g r o w i n g cells of c a p i l l a r y origin. F u r t h e r m o r e , t h e s e cells show some h i s t o c h e m i c a l c h a r a c t e r i s t i c s t y p i c a l for t h e e n d o t h e l i a l cells of b r a i n capillaries. C e r e b r a l h e m i s p h e r e s f r o m 3-day-old r a t s were dissociated in sterile c o n d i t i o n s b y p u s h i n g t h e m i n c e d b r a i n tissue t h r o u g h n y l o n sieves of 250 a n d 125 m e s h p o r e sizes. F r a g m e n t s b e i n g a t t a c h e d to t h e sieve were s u s p e n d e d in tissue c u l t u r e m e d i u m a n d p l a t e d o u t i m m e d i a t e l y o n t h e cover-slip, a c c o r d i n g to t h e c u l t u r e m e t h o d described in d e t a i l p r e v i o u s l y 4. T h e h o m o g e n a t e o b t a i n e d was c e n t r i f u g e d a n d processed f u r t h e r as d e s c r i b e d b y J o 6 a n d K a r n u s h i n a s for t h e isolation of capillaries f r o m b r a i n s of a d u l t a n i m a l s . A f t e r d i f f e r e n t i a l a n d d e n s i t y g r a d i e n t c e n t r i f u g a t i o n s , t h e p e l l e t was r e s u s p e n d e d a n d in p a r t seeded. T h e u l t r a s t r u c t u r e of t h e p e l l e t was c h e c k e d in t h e e l e c t r o n microscope (figure 1). F r o m t h e f r a g m e n t s of dissociated b r a i n tissue, as e x p e c t e d , s e v e r a l d i f f e r e n t t y p e s of ceils s t a r t e d t o grow. A m o n g t h e n e u r o n a l a n d glial cells, large ( a b o u t 25 ~ m in d i a m e t e r ) o c c a s i o n a l l y elongated, b u t as a rule r o u n d a n d f l a t ceils were growing (figure 2). T h e s a m e t y p e of cell of u n k n o w n n a t u r e was also p r e s e n t in t h o s e cultures, w h i c h were d e r i v e d f r o m t h e p e l l e t of c e n t r i f u g a t i o n s . I t was clearly

seen t h a t w h e n t h e s h o r t s e g m e n t s or longer n e t w o r k s of capillaries s e t t l e d in vitro, t h e large a n d f l a t cells o r i g i n a t e d f r o m t h e smaller, e l o n g a t e d cells of t h e c a p i l l a r y t u b e s t h e m s e l v e s (figure 3). T h e ceils of c a p i l l a r y origin, p o s s i b l y due t o t h e i r s t r o n g e r v i a b i l i t y , h a v e g r o w n f a s t e r t h a n o t h e r cells a n d w i t h i n some d a y s ( v a r y i n g f r o m 2 to 5 days) f o r m e d a c o n t i n u o u s m o n o l a y e r (figure 2). S e v e r a l n e u r o n a l a n d glial cells were o b s e r v e d t o grow l a t e r o n t h e surface of t h e m o n o l a y e r , e s t a b I i s h i n g c o n t a c t s e i t h e r w i t h e a c h o t h e r or w i t h t h e c a p i l l a r y cells. To c h a r a c t e r i z e t h e e n z y m e p a t t e r n in tile cells of c a p i l l a r y origin, h i s t o c h e m i c a l r e a c t i o n s were p e r f o r m e d . A l k a l i n e p h o s p h a t a s e s a n d d o p a - d e c a r b o x y l a s e a c t i v i t i e s c o n f i n e d to t h e c a p i l l a r y wall h a v e b e e n r e g a r d e d as c h a r a c t e r i s t i c m a r k e r e n z y m e s for t h e e n d o t h e l i a l cells of b r a i n capillaries. M a n y l a r g e a n d f l a t cells e x h i b i t e d s t r o n g a l k a l i n e p h o s p h a t a s e a c t i v i t y (figure 4), w h i c h could easily b e o b s e r v e d a m o n g t h e n o n r e a c t i v e n e u r o n a l a n d glial cells. T h e r e were, h o w e v e r , cells w h i c h did n o t s h o w a l k a l i n e p h o s p h a t a s e a c t i v i t y , a l t h o u g h t h e y h a d t h e c h a r a c t e r i s t i c s of c a p i l l a r y origin. L - d o p a was t a k e n u p to v a r y i n g e x t e n t b y a l m o s t e v e r y cell r e g a r d l e s s to its n a t u r e a n d origin (figure 5). D o p a - d e c a r b o x y l a s e , t h o u g h b e l i e v e d to i n d i c a t e a n

1 Acknowledgments. We thank Dr J. Gripenberg for technical assistance. This research was supported by Finnish Cultural Foundation and carried out during the tenure of a fellowship provided for F. J. from the Finnish-Hungarian Cultural Exchange Program. 2 Permanent address: Institute of Biophysics, Biological Research Center, Szeged, Hungary. 3 S. Varon, Neurology 48, 93 (1975). 4 H. Hervonen and L. Rechardt, Acta physiol, stand. 90, 267 (1974). 5 F. Jo6 and I. Karnushina, Cytobios 8, 41 (1973). 6 N. Shimizu, 3. comp. Neurol 93, 201 (1950).

96

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EXPERII~NTIA 34/1

i m p o r t a n t f u n c t i o n c l o s e l y r e l a t e d to t h e b l o o d b r a i n b a r r i e r of b r a i n c a p i l l a r i e s , c o u l d n o t b e u s e d i n t h e i d e n t i f i c a t i o n of cells. R o u t i n e e l e c t r o n m i c r o s c o p i c i n v e s t i g a t i o n of t h e c u l t u r e s r e v e a l e d t h a t , a m o n g o t h e r cells, t h e r e w e r e l a r g e cells s h o w i n g f e a t u r e s c h a r a c t e r i s t i c of e n d o t h e l i a l o r i g i n . D e t a i l e d d e s c r i p t i o n of t h e u l t r a s t r u c t u r a l h i s t o c h e m i c a l l o c a l i z a t i o n of t h e e n z y m e s will be given elsewhere. I t is c o n c e i v a b l e t h a t , in d i s s o c i a t e d b r a i n c u l t u r e s p r e p a r e d w i t h t h e c o n v e n t i o n a l u s e of 45 v m n y l o n s i e v e , s e g m e n t s of c a p i l l a r y t u b e s w e r e f r a g m e n t e d i n t o s m a l l e r u n i t s w h i c h c a u s e d s o m e d i f f i c u l t i e s in tile i d e n t i f i c a t i o n of t h e cells of c a p i l l a r y o r i g i n . I t m a y h a v e b e e n m i s l e a d i n g t h a t , in i m m u n o h i s t o c h e m i c a l s t u d i e s , cells w i t h m o r p h o l o g i c a l f e a t u r e s s i m i l a r t o t h e e n d o t h e l i a l cells h a v e b e e n f o u n d t o r e a c t w i t h a n t i b o d y r a i s e d a g a i n s t glial

Fig. 1. Ultrastructural view of the capillary fraction of the centrifugate. A transverse section of a capillary wall (C) is seen in the upper middle part. 2 nuclei of the endothelial cells (E) are seen. Scale bar 5 ~xm. Fig. 2. A monolayer of flat, rounded or elongated cells has formed after 12 days' culture. The cells were cultivated from the fragments harvested from the nylon mesh. Phase contrast microscopy. Scale bar 10 txm. Fig. 3. A piece of capillary tube is observed after 2 days' culture by the phase contrast microscopy. Scale bar 10 ~m.

Fig. 4. Light microscopic alkaline phosphatase activity is localized in the cytoplasm of a large, flat, most probably endothelial cell (indicated by arrows). The surrounding cells show no enzyme activity in their cytoplasm (n = nucleus). Scale bar 10 ~xm. Fig. 5. Glyoxylic acid induced fluorescence after L-dopa treatment of 14-day-old cultures. The cells originate from the fragments, which were attached to the nylon mesh. Granular fluorescence is noticed in all cultured cells. 2% glyoxylic acid treatment for 5 rain, drying and heating thereafter for 5 min at 100 ~ Scale bar 10 gm.

15. 1. 1978

Specialia

fibrillary acidic p r o t e i n a n d i n t e r p r e t e d as a s t r o c y t e s s. H o w e v e r , t h e d e m o n s t r a t i o n of capillary origin a n d t h e p r e s e n c e of m a r k e r e n z y m e , alkaline p h o s p h a t a s e , b e i n g c h a r a c t e r i s t i c of t h e e n d o t h e l i a l cells, r e n d e r it possible t h a t t h e original tissue used as a n t i g e n was in p a r t i c o n t a m i n a t e d b y capillaries. A n o t h e r possible i n t e r p r e t a t i o n could b e t h a t t h e cells g r o w i n g in c u l t u r e s w i t h v e r y similar m o r p h o l o g i c a l c h a r a c t e r i s t i c s m a y be c o m p o s e d of 2 t y p e s : one r e a c t i n g w i t h tile a n t i b o d i e s of glial fibrillary acidic p r o t e i n , a n d a n o t h e r s h o w i n g alkaline p h o s p h a t a s e p o s i t i v i t y . Before p e r f o r m i n g b i o c h e m i c a l e x p e r i m e n t s , one has to be c e r t a i n of t h e cell p o p u l a t i o n of cultures. T h e d e m o n s t r a t i o n of alkaline p h o s p h a t a s e in ceils of e n d o t h e l i a l origin m a y be of h e l p ill d e t e r m i n i n g t h e ratios b e t w e e n cells growing in vitro.

97

T h e c u r r e n t s t a t e of k n o w l e d g e of n e u r a l dissociation h a s b e e n claimed 3 to be still far f r o m s a t i s f a c t o r y . Our r e s u l t s s h o w e d t h a t , a f t e r m e c h a n i c a l dissociation of b r a i n tissue, viable cells of c a p i l l a r y origin, w h o s e n a t u r e was e v i d e n c e d as e n d o t h e l i a l cells, were p r e s e n t in t h e cultures. K e e p i n g in m i n d t h e i m p o r t a n t t r a n s p o r t p r o c e s s e s u n d e r l y i n g t h e c o m p l e x r e g u l a t o r y f u n c t i o n of t h e b l o o d b r a i n barrier, a t t e m p t s are m a d e t o o b t a i n c u l t u r e s c o n s i s t i n g m a i n l y of e n d o t h e l i a l cells in t h e h o p e of using t h e m as a n o v e l a p p r o a c h in f u r t h e r studies.

7 A. Bertler, B. Fatck, Ch. 0wman and E. Rosengren, Pharmac. Rev. 18, 369 (1966). 8 B.H. Choi and L. W. Lapham, Exp. mol. Path. 24, 110 (1976).

'Decreased serum responsiveness by'primary monolayer cultures of'preneoplastic and 'neoplastic mammary epithelial cells M. K. F e l d m a n 1

Bruce Lyon Memorial Research Laboratory, Children's Hospital Medical Center, 51st ~ Grove Streets, Oakland (California 94609, USA), 26 April 1977 Summary. M o n o l a y e r c u l t u r e s of n o r m a l , p r e n e o p l a s t i c a n d n e o p l a s t i c m u r i n e m a m m a r y epithelial cells were e x p o s e d to v a r i o u s t y p e s of m a m m a l i a n serum. A p r o g r e s s i v e decline in levels of t h y m i d i n e i n c o r p o r a t i o n t o g e t h e r w i t h a c h a n g e in t h e o r d e r i n g of sera w h i c h s t i m u l a t e s o p t i m a l i n c o r p o r a t i o n was o b s e r v e d in t h e t r a n s f o r m e d cells. M o n o l a y e r c u l t u r e s of n o r m a l m o u s e m a m m a r y epithelial cells (MMEC) r e s p o n d to t h e p r e s e n c e of s e r u m b y increasing a) t h e i r levels of D N A s y n t h e s i s 2-4, b) m i t o t i c r a t e s 5 a n d c) final cell d e n s i t i e s 6. T u m o r cells o b t a i n e d f r o m s p o n t a n e o u s l y arising (MTV-induced) m a m m a r y a d e n o c a r c i n o m a s r e s p o n d in a similar m a n n e r 7-8, H o w ever, c u l t u r e s o r i g i n a t i n g f r o m cells of t h e p r e n e o p l a s t i c ,

r--] Normal Preneoplastic ~Tumor

h y p e r p l a s t i c alveolar n o d u l e ( p r o b a b l y N I V - i n d u c e d ) h a v e n o t y e t b e e n e x a m i n e d . A l t h o u g h it is g e n e r a l l y k n o w n t h a t b o t h n o r m a l a n d a b n o r m a l m a m m a r y cells s y n t h e s i z e g r e a t e r a m o u n t s of D N A in t h e p r e s e n c e of serum, we s o u g h t here to c o m p a r e t h e degrees of r e s p o n s i v e n e s s t o v a r i o u s t y p e s of m a m m a l i a n sera u n d e r o t h e r w i s e i d e n t i c a l cell c u l t u r e conditions. The results of t h e s e e x p e r i m e n t s f o r m t h e s u b j e c t of t h i s r e p o r t . Material and methods. N o r m a l M M E C were o b t a i n e d f r o m t h e g l a n d s of 1 6 - 1 7 - d a y p r e g n a n t B A L B / c f C 3 H mice (Cancer R e s e a r c h L a b o r a t o r y , Berkeley, California). P r e n e o p l a s t i c cells were collected f r o m p r i m a r y h y p e r p l a s t i c o u t g r o w t h (HOG) of a nodule arising s p o n t a n e o u s l y in a n 8 - 1 0 - m o n t h - o l d mouse, w h e r e a s t u m o r cells were o b t a i n e d f r o m a m a m m a r y a d e n o c a r c i n o m a arising f r o m a similar HOG, b o t h f r o m B A L B / c f C 3 H mice. N o r m a l a n d p r e n e o plastic tissues were e n z y m a t i c a l l y d i s s o c i a t e d as p r e v i o u s l y d e s c r i b e d 9. N e o p l a s t i c tissue was m i n c e d a n d d i s s o c i a t e d 1

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Histogram showing levels of incorporation of aH-thymidine in the presence of various types of mammalian serum on normal, preneoplastic and neoplastic mammary epithelial cells. Each value represents the average of 4 determinations. SEM for each value is indicated by the bars.

Acknowledgments. I wish to thank Mr Donald Wong for excellent technical assistance, Ms ffolyce Hardesty for typing the final manuscript and Dr S. Abraham for reviewing the final manuscript. This work was supported by grant CA 15764 from the National Cancer Institute and grant RR 05467, Biomedical Research Support, National Institutes of Health, DHEW. 2 H.W. Hseuh and F. E. Stockdale, ~. Cell Physiol. 83, 297 (1974). 3 M . K . Feldman and D. L. Wong, Am Zool. 16, 229 (1976). 4 M. K. Feldman and D. L. Wong, In Vitro 13, 275 (1977). 5 M.K. Feldman, Ph. D. Thesis, University of California, Berkeley, California (1972). 6 M.K. Feldman, In Vitro 9, 386 (1974). 7 H.L. Hosiek and S. Nandi, Exp. Cell Res. 84, 419 (1974). 8 L . J . T . Young, R. D. Cardiff and C. M. McGrath, Tissue Cult. Ass. Manual 1, 161 (1975). 9 R.C. Foster and M. K. Feldman, Tissue Cult. Ass. Manual 1, 27 {1975). 10 T.T. Puck, S. J. Cicciura and H. W. Fisher, J. exp. Med. 106, 145 (1957). 11 R. Dulbecco and G. Freeman, Virology 8, 396 (1959).

Evidence for the presence of viable endothelial cells in cultures derived from dissociated rat brain.

15. 1. 1978 Specialia were p r e i n c u b a t e d a t 37~ for 1 min. W G A (0.01 mg] 0.1 m l of isotonic NaC1-Tris) was a d d e d to a g g l u t i...
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