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The effect of retinoic acid on the surface charge distribution in cultured human chondrosarcoma and osteosarcoma cells
Abstracts 17th Meeting Israel Society for Electron Microscopy in d i a m e t e r and w e r e u s u a l l y s t r a i g h t and non-anastomosing. We d i d not find any u l t r a s t r u c t u r a l d i f f e r e n c e s in the amyloid of two c l i n i c a l types of A m [ l o i d o s i s cutis (Lichen a m y l o i d o s i s and M a c u l a r a m y l o i d o s i s ) e x c e p t for some e p i d e r m a l changes. O u r r e s u l t s s u g g e s t that e x a m i n a t i o n by e l e c t r o n m i c r o s c o p y is r e q u i r e d for a d e f i n i t e d i a g n o s i s of A m y l o i d o s i s curls.
in the h u m a n O s t e o s a r c o m a and C h o n d r o s a r coma t u m o r cells the e x p r e s s i o n of cellular p r o p e r t i e s , w h i c h are o f t e n a s s o c i a ted w i t h the t r a n s f o r m e d p h e n o t y o e .
E L E C T R O N M I C R O S C O P Y OF IRON S T O R A G E E R Y T H R O L E U K E M I C CELLS T R E A T E D W I T H ACETYL PHENYL HYDRAZINE E. R a h a m i m , E. F i b a c h , and M. K e s s e l *
THE E F F E C T OF R E T I N O I C A C I D ON THE S U R F A C E C H A R G E D I S T R I B U T I O N IN C U L T U R E D HUMAN CHONDROSARCOMA AND OSTEOSARCOMA CELLS Y. M a r i k o v s k y ,
L. M e r o m s k y *
and R. L o t a n *
Departments of Membrane Research and *Biophysics, The W e i z m a n n I n s t i t u t e o f Science, Rehovot 76100, Israel
R e t i n o i d s , a g r o u p of n a t u r a l a n d synt h e t i c v i t a m i n A analogs, can e x e r t prof o u n d e f f e c t s on f u n d a m e n t a l c e l l u l a r p r o c e s s e s such as g r o w t h and d i f f e r e n t i a tion of normal, t r a n s f o r m e d a n d t u m o r cells in v i v o and in culture. The e f f e c t of 8 - a l l - t r a n s - r e t i n o i c a c i d (RA) on the s u r f a c e m e m b r a n e of two s a r c o m a cell lines, H s 7 9 1 d e r i v e d f r o m h u m a n O s t e o s a r coma and H s 7 0 5 d e r i v e d from h u m a n C h o n d rosarcoma, was investigated. Cell surface g l y c o p r o t e i n s w e r e l a b e l l e d on unt r e a t e d a n d on R A - t r e a t e d cells b y N a I O 4 - N a B 3 H 4, or b y n e u r a m i n i d a s e and g a l a c t o s e o x i d a s e - N a B 3 H 4. L a b e l l i n g of s e v e r a l g l y c o p r o t e i n s was e n h a n c e d 2 to 4 fold b y t r e a t m e n t w i t h i0 ~m RA. The d i s t r i b u t i o n of a n i o n i c sites on these two cell lines w a s t e s t e d w i t h c a t i o n i c f e r r i t i n (CF), a p o l y c a t i o n i c ligand. C e l l s w e r e i n c u b a t e d b r i e f l y w i t h CF, then fixed, e m b e d d e d and t h i n - s e c t i o n e d for e l e c t r o n m i c r o s c o p y (EM). Analysis by t r a n s m i s s i o n E M r e v e a l e d that CF ind u c e d a r e d i s t r i b u t i o n and g r o u p i n g of cell s u r f a c e a n i o n i c sites into c l u s t e r s and p a t c h e s , a s u r f a c e c h a r g e d i s t r i b u tion c h a r a c t e r i s t i c , w h i c h has o f t e n b e e n o b s e r v e d in v a r i o u s m a l i g n a n t t r a n s f o r m e d cell systems. Some cells e x h i b i t large portions of unlabelled surface membrane w i t h few s m a l l CF clusters, w h i l e o c c a s i o n a l m e m b r a n e p r o c e s s e s s h o w h e a v y CF patches. Treatment with RA inhibited c l u s t e r a n d p a t c h f o r m a t i o n of C F - s p e c i f ic b i n d i n g sites, thus p r o d u c i n g an e v e n ly d i s t r i b u t e d and c o n t i n u o u s CF label, as does g l u t a r a l d e h y d e f i x a t i o n p r i o r to i n c u b a t i o n of u n t r e a t e d cells w i t h CF. The r e s u l t s d e m o n s t r a t e the a b i l i t y of R A to m o d i f y the p r o d u c t i o n , g l y c o s y l a tion a n d m o b i l i t y o f cell s u r f a c e c o n s t i tuents as o b s e r v e d in EM. Our results s u p p o r t the findings that R A s u p p r e s s e s
335
IN
E. R a c h m i l e w i t z
Department of Hematology, Hadassah University Hospital, and *Department of Membrane and Ultrastructure Research, Hebrew University - Hadassah Medical School, Jerusalem 91010, Israel
M ~ s s b a u e r s p e c t r o s c o p y has d e t e r m i n e d that t r e a t m e n t of m u r i n e e r y t h r o l e u k e m i c cells (MEL) w i t h 0.01 - 0.02% a c e t y l p h e n y l h y d r a z i n e (APH) r e s u l t e d in g r a d ual d e n a t u r a t i o n of h e m o g l o b i n (Hb) and i n c o r p o r a t i o n of the r e l e a s t e H b - i r o n into ferritin. We h a v e e x a m i n e d the ult r a s t r u c t u r a l a p p e a r a n c e of i r o n - c o n t a i n ing s t r u c t u r e s in M E L c u l t u r e d in media: (a) s u p p l e m e n t e d w i t h iron c i t r a t e for s i x days, (b) w i t h the a d d i t i o n of d i m e t h y l s u l f o x i d e (DMSO) to i n d u c e e r y t h r o i d d i f f e r e n t i a t i o n , and (c) in cells o r i g i n a t i n g from this p o p u lation which were incubated with APH for 24 hrs. Previous electron microscope observations I s h o w e d that the a c c u m u l a t i o n and iron s t o r a g e in D M S O - i n c u b a t e d cells w a s f o u n d to be in d o u b l e - m e m b r a n e e n c l o s e d v e s i c l e s m e a s u r i n g 0.2 - 0.5 ~m w i t h loosely packed electron-dense particles of ferritin. Two d i f f e r e n t forms of f e r r i t i n - c o n t a i n i n g o r g a n e l l e s h a v e b e e n o b s e r v e d in the p r e s e n t study. Type I c o n t a i n s close-packed or homogeneously distributed ferrugineous material. Type II has an intralamellar appearance with evidence of l o o s e l y d i s p e r s e d g r a n u l a r content. In u n d i f f e r e n t i a t e d cells only the first type of o r g a n e l l e is p r e s e n t , w h e r e a s in the cells i n c u b a t e d w i t h D M S O alone, there is no i n d i c a t i o n o f f e r r i t i n s t r u c tures at all. H o w e v e r , in d i f f e r e n t i a t e d cells i n c u b a t e d w i t h D M S O and A P H b o t h types of s t r u c t u r e w e r e found, i n d i c a t i n g a s p e c i f i c r e a c t i o n to APH. C e l l s w e r e p r e p a r e d for e l e c t r o n m i croscopy by conventional glutaraldehydeo s m i u m t e t r o x i d e f i x a t i o n in 0.i M s a l i n e b u f f e r e d to p H 7.2. In an a t t e m p t to i d e n t i f y c l e a r l y the e l e c t r o n - d e n s e iron c l u s t e r s of ~ e r r i t i n w i t h i n the organelles s e c t i o n s w e r e not c o u n t e r - s t a i n e d w i t h u r a n y l a c e t a t e and lead citrate. i. s. ofer et al., Blood 58 (1981) 255.
in the h u m a n O s t e o s a r c o m a and C h o n d r o s a r coma t u m o r cells the e x p r e s s i o n of cellular p r o p e r t i e s , w h i c h are o f t e n a s s o c i a ted w i t h the t r a n s f o r m e d p h e n o t y o e .
E L E C T R O N M I C R O S C O P Y OF IRON S T O R A G E E R Y T H R O L E U K E M I C CELLS T R E A T E D W I T H ACETYL PHENYL HYDRAZINE E. R a h a m i m , E. F i b a c h , and M. K e s s e l *
THE E F F E C T OF R E T I N O I C A C I D ON THE S U R F A C E C H A R G E D I S T R I B U T I O N IN C U L T U R E D HUMAN CHONDROSARCOMA AND OSTEOSARCOMA CELLS Y. M a r i k o v s k y ,
L. M e r o m s k y *
and R. L o t a n *
Departments of Membrane Research and *Biophysics, The W e i z m a n n I n s t i t u t e o f Science, Rehovot 76100, Israel
R e t i n o i d s , a g r o u p of n a t u r a l a n d synt h e t i c v i t a m i n A analogs, can e x e r t prof o u n d e f f e c t s on f u n d a m e n t a l c e l l u l a r p r o c e s s e s such as g r o w t h and d i f f e r e n t i a tion of normal, t r a n s f o r m e d a n d t u m o r cells in v i v o and in culture. The e f f e c t of 8 - a l l - t r a n s - r e t i n o i c a c i d (RA) on the s u r f a c e m e m b r a n e of two s a r c o m a cell lines, H s 7 9 1 d e r i v e d f r o m h u m a n O s t e o s a r coma and H s 7 0 5 d e r i v e d from h u m a n C h o n d rosarcoma, was investigated. Cell surface g l y c o p r o t e i n s w e r e l a b e l l e d on unt r e a t e d a n d on R A - t r e a t e d cells b y N a I O 4 - N a B 3 H 4, or b y n e u r a m i n i d a s e and g a l a c t o s e o x i d a s e - N a B 3 H 4. L a b e l l i n g of s e v e r a l g l y c o p r o t e i n s was e n h a n c e d 2 to 4 fold b y t r e a t m e n t w i t h i0 ~m RA. The d i s t r i b u t i o n of a n i o n i c sites on these two cell lines w a s t e s t e d w i t h c a t i o n i c f e r r i t i n (CF), a p o l y c a t i o n i c ligand. C e l l s w e r e i n c u b a t e d b r i e f l y w i t h CF, then fixed, e m b e d d e d and t h i n - s e c t i o n e d for e l e c t r o n m i c r o s c o p y (EM). Analysis by t r a n s m i s s i o n E M r e v e a l e d that CF ind u c e d a r e d i s t r i b u t i o n and g r o u p i n g of cell s u r f a c e a n i o n i c sites into c l u s t e r s and p a t c h e s , a s u r f a c e c h a r g e d i s t r i b u tion c h a r a c t e r i s t i c , w h i c h has o f t e n b e e n o b s e r v e d in v a r i o u s m a l i g n a n t t r a n s f o r m e d cell systems. Some cells e x h i b i t large portions of unlabelled surface membrane w i t h few s m a l l CF clusters, w h i l e o c c a s i o n a l m e m b r a n e p r o c e s s e s s h o w h e a v y CF patches. Treatment with RA inhibited c l u s t e r a n d p a t c h f o r m a t i o n of C F - s p e c i f ic b i n d i n g sites, thus p r o d u c i n g an e v e n ly d i s t r i b u t e d and c o n t i n u o u s CF label, as does g l u t a r a l d e h y d e f i x a t i o n p r i o r to i n c u b a t i o n of u n t r e a t e d cells w i t h CF. The r e s u l t s d e m o n s t r a t e the a b i l i t y of R A to m o d i f y the p r o d u c t i o n , g l y c o s y l a tion a n d m o b i l i t y o f cell s u r f a c e c o n s t i tuents as o b s e r v e d in EM. Our results s u p p o r t the findings that R A s u p p r e s s e s
335
IN
E. R a c h m i l e w i t z
Department of Hematology, Hadassah University Hospital, and *Department of Membrane and Ultrastructure Research, Hebrew University - Hadassah Medical School, Jerusalem 91010, Israel
M ~ s s b a u e r s p e c t r o s c o p y has d e t e r m i n e d that t r e a t m e n t of m u r i n e e r y t h r o l e u k e m i c cells (MEL) w i t h 0.01 - 0.02% a c e t y l p h e n y l h y d r a z i n e (APH) r e s u l t e d in g r a d ual d e n a t u r a t i o n of h e m o g l o b i n (Hb) and i n c o r p o r a t i o n of the r e l e a s t e H b - i r o n into ferritin. We h a v e e x a m i n e d the ult r a s t r u c t u r a l a p p e a r a n c e of i r o n - c o n t a i n ing s t r u c t u r e s in M E L c u l t u r e d in media: (a) s u p p l e m e n t e d w i t h iron c i t r a t e for s i x days, (b) w i t h the a d d i t i o n of d i m e t h y l s u l f o x i d e (DMSO) to i n d u c e e r y t h r o i d d i f f e r e n t i a t i o n , and (c) in cells o r i g i n a t i n g from this p o p u lation which were incubated with APH for 24 hrs. Previous electron microscope observations I s h o w e d that the a c c u m u l a t i o n and iron s t o r a g e in D M S O - i n c u b a t e d cells w a s f o u n d to be in d o u b l e - m e m b r a n e e n c l o s e d v e s i c l e s m e a s u r i n g 0.2 - 0.5 ~m w i t h loosely packed electron-dense particles of ferritin. Two d i f f e r e n t forms of f e r r i t i n - c o n t a i n i n g o r g a n e l l e s h a v e b e e n o b s e r v e d in the p r e s e n t study. Type I c o n t a i n s close-packed or homogeneously distributed ferrugineous material. Type II has an intralamellar appearance with evidence of l o o s e l y d i s p e r s e d g r a n u l a r content. In u n d i f f e r e n t i a t e d cells only the first type of o r g a n e l l e is p r e s e n t , w h e r e a s in the cells i n c u b a t e d w i t h D M S O alone, there is no i n d i c a t i o n o f f e r r i t i n s t r u c tures at all. H o w e v e r , in d i f f e r e n t i a t e d cells i n c u b a t e d w i t h D M S O and A P H b o t h types of s t r u c t u r e w e r e found, i n d i c a t i n g a s p e c i f i c r e a c t i o n to APH. C e l l s w e r e p r e p a r e d for e l e c t r o n m i croscopy by conventional glutaraldehydeo s m i u m t e t r o x i d e f i x a t i o n in 0.i M s a l i n e b u f f e r e d to p H 7.2. In an a t t e m p t to i d e n t i f y c l e a r l y the e l e c t r o n - d e n s e iron c l u s t e r s of ~ e r r i t i n w i t h i n the organelles s e c t i o n s w e r e not c o u n t e r - s t a i n e d w i t h u r a n y l a c e t a t e and lead citrate. i. s. ofer et al., Blood 58 (1981) 255.